BRPI0617224B1 - RECOMBINANT DNA CONSTRUCTS - Google Patents

Abstract

METHODS FOR PRODUCING HYBRID SEED. The present invention relates to methods for producing a non-natural hybrid seed. Specific miRNAs and miRNA recognition sites useful for conferring induced sterility in a crop, and recombinant DNA construct including such exogenous miRNA recognition sites are also described.

Description

PRIORITY CLAIMS AND REFERENCE TO RELATED REQUESTS

This application claims the priority benefit of U.S. Provisional Patent Applications No. 60/726,106, filed October 13, 2005, and 60/836,246, filed August 7, 2006, which are incorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTINGS

The sequence listings contained in files "38- 21(54232)A. rpt" (file size 61 kilobytes, recorded October 12, 2005, and filed with U.S. Provisional Application 60/726,106 on October 13, 2005 ), "38-21(54232)B.rpt" (file size 68 kilobytes, recorded on August 7, 2006, and filed with U.S. Provisional Application 60/836,246 on August 7, 2006), and "38 -21(54232)C.rpt" (file size 70 kilobytes, recorded on September 19, 2006, and filed with U.S. Provisional Application xx/xxx.xxx on September 20, 2006) are incorporated herein by reference in their totalities.

FIELD OF INVENTION

The present invention relates to methods for producing hybrid seed, and inductively sterile transgenic plants and molecular constructs useful in such methods.

BACKGROUND OF THE INVENTION

Hybrid seed, that is, seed produced by hybridization or cross-fertilization of closely related plants, can be grown into progeny hybrid plants that possess "hybrid vigor" or a desirable combination of characteristics not possessed by either of their parent plants (which are typically natural plants). Hybrid plants can exhibit superior agronomic performance traits, including improved plant size, yield, nutritional composition, disease resistance, herbicide tolerance, stress tolerance (heat, cold, drought, nutrient, salt), climate adaptation, and others. desirable characteristics. Effective hybrid seed production requires that cross-pollination predominates over self-pollination. A major limitation in hybrid seed production for many crop species is the lack of simple, reliable, and economical methods of generating sterility in at least one parent (especially the male parent, to result in male-sterility while leaving the female gametes intact). and accessible for pollination by a suitable pollen donor). Male sterility is also useful where the spread of pollen is not desirable, for example, from a domestic plant to its wild relatives, or where fertilization of the flower is not desirable, for example, in the case of ornamental flowers that deteriorate in condition after pollination.

Male sterility can be achieved, for example, by physical removal of the organs containing the male gametes. In some species, this is a straightforward although laborious and therefore expensive process (e.g. detasseling in maize). In other species, such physical emasculation is difficult because of the plant's anatomy. Alternative techniques that do not involve manual or physical emasculation could provide substantial savings.

Chemical gametocides have also been described as a method of generating male-sterile plants. Typically, such a chemical gametocide is a herbicidal compound which when applied to a plant at an appropriate developmental stage or prior to sexual maturity is capable of effectively killing or exterminating the developing male gametes of a plant while leaving the female gametes, or at least a significant portion of them, capable of cross-pollination. For example, glyphosate tolerance has been genetically manipulated in corn (U.S. Patent Number 5,554,798), and use of the herbicide glyphosate (W-phosphonomethylglycine) as a gametocide, and transgenic plants that are vegetatively- and female-tolerant to glyphosate, but male-sensitive to glyphosate, is described in U.S. Patent Number 4,735,649 and PCT International Patent Application Publication WO99/46396A2. However, the levels of glyphosate required to kill the majority of male gametes while leaving a sufficient number of female gametes still capable of fertilization often resulted in plant stunting or chlorosis. Thus, a major obstacle to using glyphosate as a gametocide, as is generally true with most chemical gametocides, is the phytotoxic side effects that result from the lack of sufficient selectivity for gametes. Commercial production of hybrid seed using chemical gametocides is limited primarily by their lack of selectivity for gametes in general. Compounds that have some selectivity in reaching gametes in greater quantities than vegetative tissues are generally non-discriminative with respect to the sex of the gametes destroyed. Therefore, methods to improve the selectivity of a chemical gametocide would be highly desirable. Even more desirable would be methods that provide a first parental plant that is male-sterile, and a second parental plant that is female-sterile, thus ensuring that the seeds produced are the result of hybridization of the two parental plants, and not of self-fertilization. . .

This invention provides methods of producing hybrid seed, and further provides recombinant DNA constructs, plant chromosomes, cells, plants and transgenic seeds, which contain such constructs useful in these methods. Recombinant DNA constructs, plant chromosomes, cells, transgenic plants and seeds and methods for their use in making hybrid seed provide a greatly improved way of using herbicides like chemical gametocides. The recombinant DNA constructs of this invention include an exogenous microRNA recognition site, which allows the expression of a messenger RNA encoding a protein that confers tolerance to a herbicide to be controlled by an endogenous microRNA to a plant in which the DNA construct is present. recombinant is transcribed.

MicroRNAs (miRNAs) are non-protein-coding RNAs, generally of about 19 to about 25 nucleotides (commonly about 20 - 24 nucleotides in plants), that guide the in trans cleavage of target transcripts, negatively regulating the expression of genes involved in various regulatory and developmental pathways (Bartel (2004) Cell, 116:281-297).

In some cases, miRNAs serve to guide in-phase processing of primary siRNA transcripts (see Allen et al. (2005) Cell, 121:207-221).

Some microRNA genes (MIR genes) were identified and made publicly available in a database ("miRBase", available online at microrna.sanger.ac.uk/sequences). Applicants have described novel MIR genes, mature miRNAs, and miRNA recognition sites in U.S. Patent Application 11/303,745, filed December 15, 2005. Additional MIR genes and mature miRNAs are also described in U.S. Patent Application Publications 2005 /0120415 and 10 2005/144669A1. MIR genes have been reported to occur in intergenic regions, both isolated and in groups in the genome, but can also be located entirely or partially within introns of other genes (protein-coding or non-protein-coding). For a recent review of miRNA biogenesis, see Kim (2005) Nature Rev. Mol. Cell Biol., 6:376-385. Transcription of MIR genes may be, at least in some cases, under the promotional control of a promoter of the MIR gene itself. Transcription of the MIR gene is probably generally mediated by RNA polymerase II (see, for example, Aukerman & Sakai (2003) Plant Cell, 15:2730-2741; Parizotto et al. (2004) Genes Dev., 18:2237-2242 ), and therefore could be sensitive to gene silencing approaches that have been used on other genes transcribed by RNA polymerase II. The primary transcript. (which can be polycistronic) called a "primRNA", a miRNA precursor molecule that can be quite large (several kilobases) and contains one or more local double-stranded regions or "loop11 as well as the usual Ncap" 5' and polyadenylated tail of an mRNA. See for example, Figure 1 in Kim (2005) Nature Rev. Mol. Cell Biol., 6:376-385.

In plant cells, microRNA precursor molecules are thought to be primarily processed in the nucleus. In plants, miRNAs and siRNAs are formed by distinct DICER-like (DCL) enzymes, and in 30 Arabidopsis a nuclear DCL enzyme is believed to be required for mature miRNA formation (Xie et al. (2004) PLoS Biol., 2:642-652). Additional reviews on microRNA biogenesis and function are found, for example, in Bartel (2004) Cell, 116:281-297; Murchison & Hannon (2004) Curr. Opinion. Cell Bio!., 16:223-229; and Dugas & Bartel (2004) Curr. Opinion. Plant Biol., 7:512-520. MicroRNAs can thus be described in terms of RNA (e.g., RNA sequence of a mature miRNA or a miRNA precursor RNA molecule), or in terms of DNA (e.g., DNA sequence that corresponds to an RNA sequence of mature miRNA or a DNA sequence encoding a MIR gene or fragment of a MIR gene or a miRNA precursor). MIR gene families are estimated to be responsible for 1% of at least some genomes and capable of influencing or regulating the expression of about a third of all genes (see, for example, Tomari et al. (2005) Curr. Biol. , 15:R61-64; G. Tang (2005) Trends Biochem., 30:106-14; Because miRNAs are important regulatory elements in eukaryotes, including animals and plants, transgenic suppression of miRNAs could, for example, lead to the understanding of important biological processes or allow the manipulation of certain pathways (e.g., regulation of cell differentiation, proliferation and apoptosis) useful, for example, in biotechnological applications. See, for example, O’Donnell et al. (2005) Nature, 435:839-843; Cai et al. (2005) Proc. Natl. Academic. Know. USA, 102:5570-5575; Morris & McManus (2005) I know. STKE, pe41 (stke.sciencemag.org/cgi/reprint/sigtrans; 2005/297/pe41.pdf). MicroRNA (MIR) genes have identifying characteristics, including conservation among plant species, a stable foldback structure, and processing of a specific miRNA/miRNA* duplex by Dicer-like enzymes (Ambros etal. (2003) RNA, 9:277-279). These characteristics have been used to identify miRNAs and their corresponding genes in plants (Xie etal. (2005) Plant Physiol., 138:2145-2154; Jones-Rhoades & Bartel (2004) Mol. Cell, 14:787-799; Reinhart et al. (2002) Genes Dev., 16:1616-1626; Sunkar & Zhu (2004) Plant Cell, 16:2001-2019). Publicly available microRNA genes are cataloged in miRBase (Griffiths-Jones etal. (2003) Nucleic Acids Res., 31:439-441). MiRNAs are expressed in very specific cell types in Arabidopsis (see, for example, Kidner & Martienssen (2004) Nature, 428:8184, Millar & Gubler (2005) Plant Cell, 17:705-721). Suppression may be limited to a side, edge, or other division between cell types, and is believed to be necessary for standardization and specification of the appropriate cell type (see, e.g., Palatnik et al. (2003) Nature, 425: 257-263). Suppression of a GFP reporter gene containing an endogenous miR171 recognition site was found to limit expression to specific cells in transgenic Arabidopsis (Parízotto et al. (2004) Genes Dev., 18:2237-2242). MiRNA recognition sites were validated in all regions of an mRNA, including the 5' untranslated region, coding region, and 3' untranslated region, indicating that the position of the miRNA target site 10 relative to the coding sequence may not be necessarily affect suppression (see, for example, Jones-Rhoades & Bartel (2004). Mol. Cell, 14:787-799, Rhoades etai. (2002) Cell, 110:513-520, Allen etal. (2004) Nat Genet, 36:1282-1290, Sunkar&Zhu (2004) Plant Cell, 16:2001-2019). The mature miRNAs described here are processed from 15 MIR genes that generally belong to canonical families conserved among distantly related plant species. These MIR genes and their encoded mature miRNAs are also useful, for example, for modifying developmental pathways, for example by affecting cellular differentiation or morphogenesis (see, for example, Palatnik et al. (2003) Nature, 20 425:257 -263; Mallory et al. (2004) Curr. Biot, 14:1035-1046), to serve as sources of sequences for engineered (non-naturally occurring) miRNAs that are designed to silence sequences other than the target transcripts. naturally occurring miRNA sequence (see, e.g., Parizotto et al. (2004) Genes Dev., 18:2237-2242; see also U.S. Patent Application Publications 2004/3411A1 and 2005/0120415), and to stabilize dsRNA. A MIR gene itself (or its 5' or 3' untranslated regions, or its native promoter or other elements involved in its transcription) is useful as a target gene for gene suppression (e.g., by the methods of the present invention), where suppression of the miRNA encoded by the MIR gene is desired. MIR gene promoters can have very specific expression patterns (e.g., cell-specific, tissue-specific, or temporal-specific) and thus are useful in recombinant constructs to induce such specific transcription of a DNA sequence to which they belong. are operatively linked.

This invention provides methods for producing hybrid seed using recombinant DNA constructs including recognition sites that correspond to novel mature miRNAs that have specific expression patterns in crop plants. The recombinant DNA constructs of the invention transcribe RNA including: (a) at least one exogenous miRNA recognition site recognizable by a mature miRNA that is specifically expressed in plant reproductive tissue; and (b) messenger RNA encoding a protein that confers tolerance to an herbicide. These constructs are useful for making and using transgenic plant chromosomes, cells, plants and seeds, including inductively sterile transgenic plants, useful, for example, in hybrid seed production.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for producing a non-natural hybrid seed, including the steps of: (a) providing a transgenic, inductively sterile first parental plant that contains in its genome a recombinant DNA construct that transcribes RNA that includes : (i) at least one exogenous miRNA recognition site recognizable by a mature miRNA that is specifically expressed in reproductive tissue of the first parental plant; and (ii) a first messenger RNA encoding a protein that confers tolerance to a first herbicide, wherein the mature miRNA specifically suppresses expression of the protein in reproductive tissue, and wherein sterility of the first parental plant is inducible by application of the first herbicide the first parental plant; (b) crossing the first parental plant with a second parental plant under conditions where sterility has been induced in the first parental plant, thereby producing unnatural hybrid seed.

In another aspect, this invention provides a method for producing a hybrid seed including the steps of: (a) providing a first parental plant including a transgenic plant grown from a transgenic plant cell containing in its genome a first recombinant DNA construct that transcribes RNA that includes (i) at least one exogenous miRNA recognition site recognizable by a first mature miRNA, and (ii) a first messenger RNA that encodes a protein that confers tolerance to a first herbicide, wherein the first mature miRNA is specifically expressed in male reproductive tissue of the first parental plant, thus specifically suppressing expression of the protein in male reproductive tissue, and in which male sterility of the first parental plant is inducible by application of the first herbicide to the first parental plant; (b) providing a second parental plant that includes a transgenic plant grown from a second transgenic plant cell that contains in its genome a second recombinant DNA construct that transcribes RNA that includes (i) at least one replication site. knowledge of exogenous miRNA recognizable by a second mature miRNA, and (ii) a second messenger RNA encoding a protein that confers tolerance to a second herbicide, wherein the second mature miRNA is specifically expressed in the female reproductive tissue of the second parental plant, thus specifically suppressing expression of the protein in female reproductive tissue, and wherein female sterility of the second parental plant is inducible by application of the second herbicide to the second parental plant; (c) applying the first herbicide and the second herbicide to the parental plants, thereby inducing male sterility in the first parental plant and female sterility in the second parental plant; (d) crossing the first and second parental plants, in which ovules from the first parental plant are pollinated by pollen from the second parental plant, thus producing hybrid seed.

In an independent aspect, this invention provides an inductively sterile, transgenic plant that contains in its genome a recombinant DNA construct that transcribes RNA that includes: (a) at least one exogenous miRNA recognition site recognizable by a mature miRNA which is expressed specifically in the plant's reproductive tissue; and (b) messenger RNA encoding a protein that confers tolerance to a herbicide; in which the mature miRNA specifically suppresses the expression of the protein in reproductive tissue, and in which the sterility of the transgenic plant is inducible by application of the herbicide to the plant.

In a further aspect, this invention provides a recombinant DNA construct that transcribes RNA that includes: (a) at least one exogenous miRNA recognition site recognizable by a mature miRNA that is specifically expressed in plant reproductive tissue; and (b) messenger RNA encoding a protein that confers tolerance to a herbicide. Other specific embodiments of the invention are described in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A schematically represents non-limiting recombinant DNA constructs of the invention as described in Example 1. For use in Agrobacterium-mediated transformation of plant cells, at least one T-DNA border is generally included in each construct (not shown ). These constructs include a promoter ("pro") element, an intron flanked on one or both sides by non-protein-coding DNA, an optional terminator ("ter") element, at least one first gene suppression element ( "GSE" or "GSE1") to suppress at least one first target gene, and may optionally include at least one second gene suppression element ("GSE2") for suppressing at least one second target gene, at least one expression element 20 gene ("GEE") to express at least one gene of interest, or both.

In embodiments that contain a gene expression element, the gene expression element may be located adjacent to (or outside of) the intron. In a variation of this embodiment (not shown), the gene suppression element (inserted into an intron flanked on one or both sides by non-protein-coding DNA) is located 3' to the terminator. In other constructs of the invention (not shown), a gene suppression element (not inserted into the intron) is located 3' to the terminator (see Example 22). Figure 1B schematically represents examples of DNA constructs distinct from those of the present invention. Such constructs may contain a gene suppression element that is located adjacent to an intron or between two distinct introns (that is, not inserted within a single intron), or may include a gene expression element that includes a suppression element. gene inserted within an intron that is flanked on both sides by protein-coding DNA (e.g., protein-coding exons that form a gene expression element). Figure 2 depicts several non-limiting examples of gene suppression elements and exogenous DNAs capable of being usefully transcribed into the recombinant DNA constructs of the invention as described in Example 1. Where drawn as a single strand (Figures 2A through 2E), these are conveniently represented in the 5' to 3' (left to right) transcriptional direction, where arrows indicate anti-sense sequence (arrowhead pointing to the left), or sense sequence (arrowhead pointing to the right). Where drawn as double-stranded (antiparallel) transcripts (Figures 2F and 2G), 5' and 3' transcriptional directionality is shown. Solid lines, dashed lines, and dotted lines indicate sequences that hit different target genes. Such gene suppression elements and exogenous DNAs capable of being transcribed may include: DNA that includes at least one segment of antisense DNA that is antisense to at least one segment of the first target gene, or DNA that includes multiple copies of at least one antisense DNA segment that is antisense to at least one segment of at least one first target gene (Figure 2A); DNA that includes at least one segment of sense DNA that is at least one segment of the at least one first target gene, or DNA that includes multiple copies of at least one segment of sense DNA that is at least one segment of the at least one first gene target (Figure 2B); DNA that transcribes RNA 25 to suppress the at least one first target gene by forming double-stranded RNA and includes at least one antisense DNA segment that is antisense to at least one segment of the at least one target gene and at least a sense DNA segment that is at least a segment of the at least one first target DNA (Figure 2C); DNA that transcribes RNA 30 to suppress the at least one first target gene by forming a single double-stranded RNA and includes multiple serial antisense DNA segments that are antisense to at least one first target gene and multiple sense DNA segments serials that are at least a segment of at least one first target gene (Figure 2D); DNA that transcribes RNA to suppress the at least one first target gene by forming multiple double-stranded RNA and includes multiple antisense DNA segments that are antisense to at least one segment of the at least one first target gene and multiple segments of sense DNA which are at least one segment of the at least one first target gene, and wherein said multiple segments of antisense DNA and the multiple segments of sense DNA are arranged in a series of inverted repeats (Figure 2E); and DNA that includes nucleotides derived from a miRNA, or DNA that includes nucleotides from an siRNA (Figure 2F). Figure 2F depicts various non-limiting double-stranded RNA (dsRNA) arrays that can be transcribed from embodiments of the gene suppression elements and exogenous DNAs capable of being usefully transcribed into the recombinant DNA constructs of the invention. When such dsRNA is formed, it can suppress one or more target genes, and can form a single double-stranded RNA or multiple double-stranded RNA, or a single stem or multiple stems of dsRNA. Where multiple dsRNA "stems" are formed, they may be arranged in "hammerhead" or "cloverleaf" arrangements. Spacer DNA is optional and may include sequence that transcribes an RNA (e.g., a large sequence loop). antisense of the target gene or an aptamer) that assumes a secondary structure or three-dimensional configuration that confers on the transcript a desired characteristic, such as increased stability, increased in vivo half-life, or cell or tissue specificity. expression of the indicated mature miRNAs in various maize tissues, as described in detail in Example 2. Figure 4 represents a non-limiting example of a DNA sequence capable of being transcribed that includes an exogenous miRNA recognition site, chloroplast-targeted TIC809 with a miRNA162 recognition site (bold text) located in the 3' untranslated region (SEQ ID NO. 176), as described in detail in Example 3. The translated amino acid sequence is also shown. Figure 5 represents a non-limiting example of a DNA sequence capable of being transcribed that includes an exogenous miRNA recognition site, non-chloroplast-targeted TIC809 with a miRNA164 recognition site (bold text) located in the 3' untranslated region ( SEQ ID NO. 177), as described in detail in Example 3. The translated amino acid sequence is also shown. Figure 6 depicts the strong and specific endosperm expression of the microRNA miR167g (SEQ ID NO. 178) cloned from maize endosperm, as described in detail in Example 4. Northern blots of RNA from maize tissues (LH59) labeled with a end-labeled mature miR167 22-mer LNA probe specific for SEQ ID NO. 178 (Figure 6A) or with a ~400bp miR167g-specific gene probe (Figure 6B). Transcriptional profiling of maize tissues confirmed the Northern blot results (Figure 6C); the transcript corresponding to miR167g was abundantly and specifically expressed in endosperm tissue (abundances are categorized as follows: >5000, high abundance, 97th percentile; 700 - 5000, moderate abundance, 20th percentile; 400 - 700, medium abundance; 200 - 400, low abundance; <200, not detected). Selected abbreviations: "DAP" or "DA", days after pollination; "WK", whole grain, "endo", endosperm. Figure 7 represents a partial annotation map, including the gene locations of mIR167a and miR167g and mature miRNAs, and promoter elements (e.g., TATA boxes), of the genomic cluster within which the miR167g promoter sequences were identified as described in detail in Example 4. Abbreviations: "PBF", "prolamin box" binding factor; "ARF“ auxin-responsive factor (auxin binding); "NIT2", activator of nitrogen-related genes; "LYS14", element that binds to UASLYS, an upstream activating element that confers apparent Lys14-dependent activation and repression and adipate semialdehyde; "GLN3", an element that binds nitrogen upstream of the glutamine synthetase activation sequence. Figure 8 represents results described in detail in Example 5. Figure 8A represents the "fold-back" structure of SEQ ID. NO. 186, the predicted miRNA precursor for SEQ ID NO. 184; the mature miRNA is located at bases 106 - 126, the corresponding miRNA* at bases 156 - 175, and another abundant miRNA was found to be located at bases 100 - 120 in the stem of the fold-back structure. "Count" refers to the number of occurrences of a small RNA in the filtered group of 381,633 putative miRNA sequences that were analyzed. the miRNA precursor SEQ ID NO. 186. Figure 8C depicts a transcriptional profile in soybean tissues for a predicted target, polyphenol oxidase (SEQ ID NO. 200) for the mature miRNA (SEQ ID NO. 184). Figure 9 represents the results described in detail in Example 5. Figure 9A represents the fold-back structure of SEQ ID NO. 189, the predicted miRNA precursor for SEQ ID NO. 187; the mature miRNA is located at bases 163 - 183, and the miRNA* at bases 18-63. “Count” refers to the number of occurrences of a small RNA in the filtered group of 381,633 putative miRNA sequences that were analyzed. Figure 9B depicts a transcriptional profile in soybean tissues for a predicted target, polyphenol oxidase (SEQ ID NO. 251) for the mature miRNA (SEQ ID NO. 187). Figure 10 represents the results described in detail in Example 5. Figure 10A represents the fold-back structure of SEQ ID NO. 192, the predicted miRNA precursor for SEQ ID NO. 190; the mature miRNA is located at bases 87 - 107, and the miRNA* at bases 150 - 169. "Count" refers to the number of occurrences of a small RNA in the filtered group of 381,633 putative miRNA sequences that were analyzed. Figure 11 represents the results described in detail in Example 5. Figure 11A represents the fold-back structure of SEQ ID NO. 195, the predicted miRNA precursor for SEQ ID NO. 193; the mature miRNA is located at bases 61 - 81, and the miRNA* at bases 109 - 129. "Count" refers to the number of small RNA hits in the filtered group of 381,633 putative miRNA sequences that were analyzed. Figure 12 represents the results described in detail in Example 5. Figure 12A (top) represents the fold-back structure of SEQ ID NO. 198, one of the predicted miRNA precursors for SEQ ID NO. 196; the mature miRNA is located at bases 157 - 178, and the miRNA* at bases 72 - 93. Figure 12A (bottom) represents the "ufold-back" structure of SEQ ID NO. 199, another predicted miRNA precursor for SEQ ID NO. .196; the mature miRNA is located at bases 123 - 144, and the miRNA* at 5 bases 58 - 79. “Count” refers to the number of occurrences of a small RNA in the filtered group of 381,633 putative miRNA sequences that were selected. analyzed. Figure 12B (top) represents a transcriptional profile in soybean tissues for the precursor miRNA SEQ ID NO. 198. Figure 12B (bottom) depicts a transcriptional profile in soybean tissues for the precursor miRNA SEQ ID NO. 199. Figures 13 and 14 represent the transcriptional profiles of probe sets of sequences that have been identified as including miRNA hl genes (given in Table 6) especially useful in methods of this invention and that have the desired expression patterns, i.e. in female reproductive tissue (Figure 13) or in male reproductive tissue (Figure 14), as described in detail in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used and the manufacturing or laboratory procedures described below are well-known and commonly employed in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references. Unless otherwise stated, nucleic acid sequences in the text of this application are given, when read from left to right, in the 5' or 3' direction. Where a term is given in the singular, inventors also contemplate aspects of the invention described by the plural of that term. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in that application shall have the definitions given. Other technical terms used have their common meaning in the technique in which they are used, as exemplified by a variety of technical dictionaries. Inventors do not intend to be limited to one mechanism or mode of action. Reference to this is provided for illustrative purposes only.

METHOD FOR PRODUCING NON-NATURAL HYBRID SIMILAR

A first aspect of this invention provides a method for producing a non-natural hybrid seed, including the steps of: (a) providing a first transgenic, inductively sterile parental plant that contains in its genome a recombinant DNA construct that transcribes RNA that includes: ( i) at least one exogenous miRNA recognition site recognizable by a mature miRNA that is specifically expressed in reproductive tissue of the first parental plant; and (ii) a first messenger RNA encoding a protein that confers tolerance to a first herbicide, wherein the mature miRNA specifically suppresses expression of the protein in reproductive tissue, and wherein sterility of the first parental plant is inducible by application of the first herbicide the first parental plant; (b) crossing the first parental plant with a second parental plant under conditions where sterility has been induced in the first parental plant, thereby producing unnatural hybrid seed. Plants: The method of this invention includes providing an inductively sterile, transgenic plant useful as a parent plant, which is preferably a seed-propagated crop plant, such as those described under the heading "Making and Using Transgenic Plant Cells and Transgenic Plants”. Non-limiting examples of preferred plants include corn, rice, wheat, oats, barley, rye, triticale, millet, sorghum, quinoa, amaranth, buckwheat, forage grass, peat, alfalfa, cotton, safflower, sunflower, soybeans, canola, rapeseed, flax, peanuts, beans, peas, lentils, alfalfa, lettuce, asparagus, artichokes, celery, carrots, radishes, cabbage, kale, mustard, broccoli, cauliflower, Brussels sprouts, turnips, kohlrabi, cucumbers, melons , strawberry pumpkin, pumpkin, onion, leek, shallots, chives, tomato, eggplant, pepper, physalis, beet, chard, spinach, ornamental plants, and forest trees. Particularly preferred plants include corn, rice, wheat, cotton, safflower, sunflower, canola, and rapeseed. In a particularly preferred embodiment, the method is useful for producing non-natural hybrid corn seed.

Sterility: By "sterility" is meant the inability to successfully produce viable progeny seed. Male sterility includes, for example, the inability to produce male gametes (e.g., fertile, mature pollen grains) capable of fertilizing female gametes, or 5 the inability to produce male reproductive structures, (e.g., anthers, stamens, filaments ) necessary for male gametes to be capable of normal fertilization of female gametes. Female sterility includes, for example, the inability to produce female gametes capable of being fertilized, or the inability to produce a fully developed seed (whether viable or sterile), or the inability to produce viable progeny seed capable of being germinated. and develop into a normal progeny plant. Reproductive tissue: in embodiments of the inductively sterile transgenic plant, where sterility is male sterility, the reproductive tissue is male reproductive tissue. Male reproductive tissue includes any tissue that is necessary for the development of male gametes for fertility (i.e., that has the ability to fertilize a female gamete), e.g. pollen or male gametes at any stage of development, anthers, stamens, fillet , tassels, and other male floral parts, nutritive tissues, and other structures involved in the ability of a male parent plant to fertilize a female parent plant.

In embodiments of the inductively sterile transgenic plant where the sterility is female sterility, the reproductive tissue is female reproductive tissue. Female reproductive tissue includes any tissue that is necessary for the development of female gametes for fertility (i.e., that have the capacity to be fertilized by a male gamete, more preferably that have the capacity to produce a fully developed seed (whether viable or sterile) ), and in many embodiments most preferably having the ability to produce viable progeny seed capable of being germinated and developed into a normal progeny plant). Female reproductive tissue includes, for example, female ovules or gametes at any stage of development, stigmas, pistils, and other female floral parts, seed coats, fruits, rachises, ears, or other nutritive, supportive, or protective tissues, and any other structure involved in the ability of a female parent plant to be fertilized by a male parent plant, produce a fully developed seed, or produce a viable progeny seed. In certain embodiments, sterility results in the failure of a fertilized egg to develop into a viable seed, and "reproductive tissue" refers to a fertilized egg (e.g., a zygote or an embryo of any stage of development, or the maternal tissues necessary for the development of a fertilized egg into a seed such as an endosperm or other nutritive tissues). mature miRNA: by "mature miRNA" is meant the small RNA ("microRNA") processed from a miRNA precursor (e.g. primRNA or pre-miRNA), which is capable of recognizing and binding to a specific sequence ("miRNA recognition site") within an RNA transcript, and guide the cleavage of that transcript. Preferred mature miRNAs include mature miRNAs that are specifically expressed in the reproductive tissue of a plant. In a non-limiting embodiment of the invention, the mature miRNA is a miRNA from a crop plant, such as a corn miRNA or a soybean miRNA. Non-limiting examples of miRNAs useful in this invention are provided in the Working Examples and include, but are not limited to, the miRNAs identified in Tables 1, 2, 3, 4, 5 and 6, including the miRNAs that have SEQ ID NO. 297, SEQ ID NO. 302, SEQ ID NO. 314, SEQ ID NO. 319, SEQ ID NO. 323, SEQ ID NO. 328, SEQ ID NO. 333, or SEQ ID NO. 338. MiRNA recognition sites: The recombinant DNA construct transcribes RNA that includes one or more exogenous miRNA recognition sites, which may be one or more copies of the same exogenous miRNA recognition site or one or more copies of different exogenous miRNA recognition sites. The exogenous miRNA recognition site can be located at any location on the transcribed RNA where, when the mature miRNA is not present, transcription produces a messenger RNA that is translatable to the desired herbicide tolerance protein, and where the mature miRNA is present, the mature miRNA binds to the RNA transcript and suppresses the expression of messenger RNA. One or more exogenous miRNA recognition sites may be located in coding regions or non-coding regions of the messenger RNA. In various embodiments, the exogenous miRNA recognition site is located within at least one of (a) a region 5' of the messenger RNA coding sequence; (b) a region 3' of the messenger RNA coding sequence; and (c) messenger RNA. In a preferred embodiment, the at least one miRNA recognition site is located in the 3' untranslated region of the messenger RNA. In various embodiments, the recombinant DNA construct further includes at least one element selected from: (a) a plant promoter; (b) a gene suppression element; (c) an intron; (d) a gene expression element; (e) DNA that transcribes an RNA aptamer capable of binding a ligand; (f) DNA that transcribes an RNA aptamer capable of binding a ligand and DNA that transcribes regulatory RNA capable of regulating the expression of a target sequence, characterized in that regulation is dependent on the conformation of the regulatory RNA, and the conformation of the Regulatory RNA is affected allosterically by the binding state of the RNA aptamer, and (g) at least one T-DNA edge. These additional elements are described in detail below under the heading "Inductively Sterile Transgenic Plants Containing a Recombinant DNA Construct".

Any miRNA recognition site that is recognized by a mature miRNA that has the desired expression pattern (i.e., specific expression in male or female reproductive tissue) is useful in embodiments of this invention. Multiple miRNA recognition sites can be used to specifically suppress protein expression in a desired combination of reproductive tissues. For example, a recombinant DNA construct may include multiple recognition sites, each of which is recognized by a miRNA that has specific expression in a different male (or female) reproductive tissue (e.g., a miRNA expressed in pollen and a miRNA expressed in the tassel). The selection of an appropriate mature miRNA or its corresponding miRNA recognition site is carried out by methods known to those skilled in the art, including those illustrated in detail in the working examples provided here. Methods include cloning mature miRNA molecules or miRNA precursors (pre-miRNAs, primRNAs) from whole plants or seeds or from selected tissues, bioinformatics-based miRNA information, Southern blots of miRNA or miRNA precursor molecules, determining the patterns of miRNA promoter expression, etc. In non-limiting embodiments, the miRNA recognition site is recognized by a mature miRNA derived from the fold-back11 structure of a MIR sequence identified in Tables 1 to 6; Particularly preferred are the miRNA recognition sites recognized by the mature miRNAs identified in Table 6.

Cleavage of a target RNA transcript and subsequent suppression of the target RNA is dependent on base pairing between the mature miRNA and its cognate miRNA recognition site. In this way, the at least one exogenous miRNA recognition site is designed to have sufficient sequence complementarity to the mature miRNA to allow recognition and binding by the mature miRNA. In plants, sequence complementarity of a miRNA and its recognition site is typically high, for example, perfect complementarity between 19, 20 or 21 of 22 nucleotides (in the case of a mature miRNA, which is 21 nucleotides long), that is, complementarity of about 90% or more. A similar degree of complementarity is preferable for recognition sites for plant miRNAs of any length (e.g., 20, 21, 22, 23, and 24 nucleotides). The sequence requirements for mature miRNA binding to a recognition site, and methods for predicting miRNA binding to a given sequence, are discussed, for example, in Llave et al. (2002) Science, 297:2053-2056, Rhoades et al. (2002) Cell, 110:513-520, Jones-Rhoades and Bartel (2004) Mol. Cell, 14:787-799, Schwab etal (2005) Developmental Cell, 8:517-527, and Xie etal. (2005) Plant Physiol., 138:21452154. When designing a miRNA recognition site as well as its exact location in, or adjacent to, a messenger RNA, it is also preferable to avoid sequences that have undesirable characteristics, such sequences encoding undesirable polypeptides, as described below under the heading "Genes Target". By designing messenger RNA as a transgene to be expressed, the unintentional introduction of an exogenous miRNA recognition site is avoided where suppression by a mature miRNA is not desired.

Specific expression: By "specifically expressed in reproductive tissue" it is meant that the mature miRNA is transcribed in reproductive tissue at a level sufficient to allow the miRNA to specifically suppress expression of the protein in reproductive tissue, i.e., specifically suppress the expression in reproductive tissue of the protein that confers tolerance to a herbicide, but does not substantially suppress expression of the same protein in tissues other than reproductive tissues. Substantial suppression of expression includes at least 30, at least 50, at least 60, at least 70, at least 80, at least 85, at least 90, 15 at least 95, or at least 98 percent suppression of expression, in relationship to that in control cells (i.e., cells that include an analogous recombinant DNA construct, e.g., a recombinant DNA construct that transcribes RNA that includes the same messenger RNA that encodes a protein that confers tolerance to a herbicide , but which does not include an exogenous RNA recognition site recognizable by the mature miRNA specifically expressed in plant reproductive tissue). In preferred embodiments, the mature miRNA specifically suppresses expression of the protein in reproductive tissue to a degree sufficient to result in sterility of the reproductive tissue when the herbicide is applied.

Those skilled in the art recognize that, in certain cases, tolerance to a herbicide can be adequately obtained at moderate or even low expression levels of a herbicide tolerance protein. Therefore, it is not a requirement that the mature miRNA be completely absent in tissues other than reproductive tissue, nor is it a requirement that herbicide tolerance protein expression be completely suppressed in reproductive tissue. Protein that confers tolerance to herbicide: any protein that confers tolerance to a plant to an herbicide can be used. In many embodiments, the herbicide is a systemic herbicide. Non-limiting examples of herbicides useful in aspects of this invention include the herbicides glyphosate, dicamba, glufosinate, sulfonylureas, imidazolinones, bromoxynil, dalapon, dicamba, cycloezanedione, protoporphyrinogen oxidase inhibitors, and isoxaflutole. Non-limiting examples of proteins that confer tolerance to glyphosate include 5-enolpyruvylshikimate-3-phosphate synthase ("EPSPS" or "aroA", see U.S. Patent Nos. 5,627,061, 5,633,435, 6,040,497, and 5,094,945) , glyphosate oxidoreductase ("GOX", see U.S. Patent Number 5,463,175), glyphosate decarboxylase (see U.S. Patent Application Publication 2004/0177399), glyphosate/V-acetyl transferase ("GAT", see U.S. Patent Application Publication 2003/0083480) or any other gene that provides resistance to glyphosate. A particularly preferred embodiment is "epsps-cp4" or 5-enolpyruvylshikimate-3-phosphate synthase from the CP4 strain of Agrobacterium tumefaciens; nucleic acid and amino acid sequences of epsps-cp4, transformation vectors that contain this gene, and methods of making plants resistant to glyphosate. Non-limiting examples of proteins that confer tolerance to other herbicides include dicamba monooxygenase, which confers tolerance to auxin-like herbicides. such as dicamba (see U.S. Patent Application Publications 2003/0115626, 2003/0135879); phosphinothricin acetyltransferase ("pat" or "bar1'), which confers tolerance to phosphinothricin or glufosinate (see U.S. Patent Numbers 5,646,024, 5,561,236, 5,276,268, 5,637,489, and US 5,273,894; see also European Patent Application EP 275,957;); 2,2-dichloropropionic acid dehalogenase, which confers tolerance to 2,2-dichloropropionic acid ("Dalapon") (see PCT Patent Publication WO99/27116); which confers tolerance to acetolactate synthase inhibitors such as sulfonylurea, imidazoinone, triazolopyrimidine, pyrimidyloxybenzoates, and phthalide (see U.S. Patent Nos. 6,225,105, 5,767,366, 4,761,373, 5,633,437, 6,613,963, 59 , 5,141,870, 5,378,824 and 5,605,011); haloarylnitrilase ("Bxn"), which confers tolerance to bromoxynil (see U.S. Patent number 4,810,648; see also PCT Patent Application Publications W089/00193 and WQ87/04181A1 ); acetyl-coenzyme A modified carboxylase, which confers tolerance to cyclohexanedione ("sethoxydim") and aryloxyphenoxypropionate ("haloxyfop") (see U.S. Patent number 6,414,222); dihydropteroate synthase ("sul I"), which confers tolerance to sulfonamide herbicides (see U.S. Patent Numbers 5,597,717, 5,633,444, and 5,719,046); 32 kDa photo-5 system II polypeptide ("psbA1'), which confers tolerance to triazine herbicides (see Hirschberg etal. (1983) Science, 222:1346-1349); anthranilate synthase, which confers tolerance to 5-methyltryptophan (see U.S. Patent number 4,581,847); dihydrodipicolinic acid synthase (“dap A”), which confers tolerance to aminoethyl cysteine (see PCT Patent Application Publication 10 WO89/11789); phytoene desaturase ("crtl"), which confers tolerance to pyridazinone herbicides such as norflurazon (see Japanese Patent Publication JP06343473); hydroxyphenyl pyruvate dioxygenase, which confers tolerance to cyclopropyl isoxazole herbicides such as isoxaflutole (see U.S. Patent Number 6,268,549; see also PCT Patent Application Publication 15 WO96/38567); modified protoporphyrinogen oxidase I ("protox"), which confers tolerance to protoporphyrinogen oxidase inhibitors (see U.S. Patent number 5,939,602); aryloxyalkanoate dioxygenase ("AAD-1"), which confers tolerance to a herbicide containing an aryloxyalkanoate moiety (see PCT Patent Application Publication WO05/107437), examples of such herbicides include phenoxy auxins (such as 2, 4-D and dichlorprop), pyridyloxy auxins (such as fluroxypyr and triclopyr), aryloxyphenoxypropionate (AOPP) acetylcoenzyme A carboxylase (ACCase) inhibitors (such as haloxyfop, quizalofop, and diclofop) and phenoxyacetate protoporphyrinogen oxidase IX 5- modified inhibitors (such as pyraflufen and flumiclorac).

Parental Plant Combinations: The method of this invention can be carried out using various combinations of the first parent plants and second parent plants. In many embodiments of the method, the second parental plant is tolerant to the first herbicide; for example, the second parental plant may contain in its genome a transgene that confers tolerance to the first herbicide. This invention encompasses embodiments of the method in which: (a) the reproductive tissue is male reproductive tissue, the first parental plant is inductively male sterile, and the second parental plant is normally fertile; or (b) the reproductive tissue is male reproductive tissue, the first parental plant is inductively male-sterile, and the second parental plant is female-sterile; or (c) the reproductive tissue is male reproductive tissue, the first parental plant is inductively male-sterile, and the second parental plant is inductively female-sterile; or (d) the reproductive tissue is female reproductive tissue, the first parental plant is inductively female-sterile, and the second parental plant is normally fertile; or (e) the reproductive tissue is female reproductive tissue, the first parental plant is inductively female-sterile, and the second parental plant is male sterile; or (f) the reproductive tissue is female reproductive tissue, the first parental plant is inductively female-sterile, and the second parental plant is inductively male-sterile.

In one embodiment of the method, the reproductive tissue is male reproductive tissue, the first parental plant is inductively male-sterile, and the second parental plant includes a second transgenic, inductively female-sterile parental plant containing in its genome a DNA construct. recombinant that transcribes RNA that includes: (i) at least one exogenous miRNA recognition site recognizable by a second mature miRNA that is specifically expressed in female reproductive tissue of the second parental plant; and (ii) a second messenger RNA 20 encoding a second protein that confers tolerance to a second herbicide; wherein the second mature miRNA specifically suppresses expression of the second protein in female reproductive tissue, and wherein female sterility of the second parental plant is inducible by application of the second herbicide to the second parental plant. In another embodiment, the reproductive tissue 25 is female reproductive tissue, the first parental plant is inductively female sterile, and the second parental plant includes a second transgenic, inductively male sterile parental plant that contains in its genome a recombinant DNA construct that transcribes RNA which includes: (i) at least one exogenous miRNA recognition site recognizable by a second mature miRNA that is specifically expressed in male reproductive tissue of the second parental plant; and (ii) a second messenger RNA that encodes a second protein that confers tolerance to a second herbicide, wherein the second mature miRNA specifically suppresses expression of the second protein in male reproductive tissue, and wherein the male sterility of the second plant parental is inducible by application of the second herbicide to the second parental plant. In these embodiments, the first and second messenger RNAs may be identical, or 5 may be different. In these embodiments, the first herbicide and the second herbicide may be identical or may be different. In some cases, . at least one of the first herbicide and the second herbicide includes a systemic herbicide. In preferred embodiments, at least one of the first herbicide and the second herbicide includes at least one herbicide selected from the group consisting of glyphosate, dicamba, glufosinate, sulfonylureas, imidazolinones, bromoxynil, 2,2-dichloropropionic acid. , acetolactate synthase inhibitors, cycloezanedione, aryloxyphenoxypropionate, sulfonamide herbicides, triazine herbicides, 5-methyltryptophan, aminoethyl cysteine, pyridazinone herbicides, cyclopropylisoxazole herbicides, pro-toporphyrinogen oxidase inhibitors, and herbicides that contain a portion of aryloxyalkanoate. .

In one embodiment of the method, the first parental plant is an inductively sterile transgenic plant that contains in its genome a recombinant DNA construct that transcribes RNA that includes: (i) at least one exogenous miRNA recognition site recognizable by a mature miRNA which is specifically expressed in male reproductive tissue of the plant; and (ii) messenger RNA encoding a protein that confers tolerance to a herbicide, in which the mature miRNA specifically suppresses expression of the protein in male reproductive tissue, and in which male sterility of the transgenic plant is inducible by application of the herbicide to the plant, and the second parental plant is normally fertile. To induce male sterility of the first parental plant, the herbicide is preferably applied under a regimen that results in the inability of the first parental plant to produce male gametes (or male reproductive structures) necessary for normal fertilization of the second parental plant. In such embodiments, the second parental plant is generally tolerant to the same herbicide used to induce male sterility as the first parental plant, and thus the first and second parental plants can be treated with the same herbicide, preferably simultaneously (e.g., together in a field). ). A cross of the first and second parent plants produces seed that is the result of pollination of the first parent plant by the second parent plant.

In another embodiment of the method, the first parental plant is an inductively sterile transgenic plant that contains in its genome a recombinant DNA construct that transcribes RNA that includes: (i) at least one exogenous miRNA recognition site recognizable by a mature miRNA; and (ii) a messenger RNA encoding a protein that confers tolerance to a herbicide, wherein the mature miRNA specifically suppresses expression of the protein in female reproductive tissue or in the fertilized egg, and wherein the female sterility of the transgenic plant is inducible by application of the herbicide to the plant, the herbicide is preferably applied under a regime that results, for example, in the inability of the first parental plant to produce female gametes capable of being fertilized, or in the inability to produce a fully developed seed (either viable or sterile), or the inability to produce viable progeny seed capable of being germinated and developing into a normal progeny plant. In such embodiments, the second parental plant is generally tolerant to the same herbicide used to induce female sterility as the first parental plant, and thus the first and second parental plants can be treated with the same herbicide, preferably simultaneously. A cross of the first and second parent plants produces seed that is the result of pollination of the second parent plant by the first parent plant. Another aspect of the invention provides a method for producing a non-natural hybrid seed, which includes the steps of: (a) providing a first parental plant that includes a transgenic plant grown from a first transgenic plant cell (e.g., a first transgenic corn cell) that contains in its genome a first recombinant DNA construct that transcribes RNA that includes: (i) at least one exogenous miRNA recognition site recognizable by a first mature miRNA, and (ii) a first RNA messenger encoding a protein that confers tolerance to a first herbicide, wherein the first mature miRNA is specifically expressed in male reproductive tissue of the first parental plant, thereby specifically suppressing expression of the protein in male reproductive tissue, and wherein male sterility of the first parental plant is inducible by applying the first herbicide to the first parental plant; (b) providing a second parental plant that includes a transgenic plant grown from a second transgenic plant cell (e.g., a second transgenic corn cell) that contains in its genome a second recombinant DNA construct that transcribes RNA that includes : (i) at least one exogenous miRNA recognition site recognizable by a second mature miRNA, and (ii) a first messenger RNA encoding a protein that confers tolerance to (| a second herbicide, wherein the second mature miRNA is specifically expressed in female reproductive tissue of the second parental plant, thereby specifically suppressing expression of the protein in female reproductive tissue, and wherein female sterility of the second parental plant is inducible by applying the second herbicide to the second parental plant; the first herbicide and the second herbicide to the parental plants, thereby inducing male sterility in the first parental plant and female sterility in the second parental plant; (d) crossing the first and second parental plants, in which ovules from the first parental plant are pollinated by pollen from the second parental plant, thus producing unnatural hybrid seed (e.g., unnatural hybrid corn seed).

When practicing any of the methods for producing hybrid seed, "cultivated" refers to cultivating a transgenic plant by direct regeneration from the first transgenic plant cell or cultivating a transgenic progeny plant from the regenerated transgenic plant. Methods for directly regenerating a transgenic plant from a transgenic plant cell, or for cultivating transgenic progeny plants (including natural progeny plants and hybrid progeny plants) are well known in the art, and are described herein under the title "Making and Using Transgenic Plant Cells and Transgenic Plants". The second messenger RNA may be the same or may be different from the first messenger RNA. In various embodiments, the first

Messenger RNA and the second messenger RNA (a) are identical; or (b) are different and encode different segments of the protein; (c) are different and encode different protein segments. In preferred embodiments, the first and second messenger RNA (whether the same or different) confer tolerance to the same herbicide, which provides the convenience of treating a field of the first and second parental plants mixed with a single herbicide; When necessary, the herbicide is applied at one or more times in order to induce male sterility of the first parental plant and female sterility of the second parental plant. In some cases, the first messenger RNA encodes a first protein that confers tolerance to a herbicide, and the second messenger RNA encodes a second, different protein that confers tolerance to the same herbicide; A non-limiting example is where the first and second proteins are selected from a group (including 5-ene1pyruvylshikimate-3-phosphate synthase, glyphosate acetyltransferrase and glyphosate decarboxylase) which all confer tolerance to glyphosate. In other embodiments, the first and second messenger RNAs confer tolerance to two different herbicides, and preferably the first and second parental plants both contain in their genome DNA encoding two proteins, each conferring tolerance to one of the two herbicides. , allowing a field of the first and second parental plants to be treated with both herbicides (simultaneously or separately). In various embodiments of the method the first herbicide and the second herbicide (a) are identical; (b) are different (wherein the first and second parental plants preferably have tolerance to both the first and second herbicide).

Herbicide application regimes are used whereby inducible sterility is obtained preferably without field loss or other undesirable results. Herbicide application(s) are regulated in order to induce sterility. In an analogous example, a transgenic cotton line (RR1445) that contains EPSPS under the control of a constitutive FMV 35S promoter, was found to have high expression of EPSPS in most vegetative tissues, but low expression of EPSPS in certain male reproductive tissue types (pollen mother cells, male gametophytes, and tapetum); Applications of glyphosate after the four-leaf stage result in male sterility (see, Chen et al. (2006) Plant Biotechnol. J., 4:477-487, published in advance online June 7, 2006, doi: 10.1111 /j. 1467-7652.2006.00203.x). ,

In preferred embodiments, the method is useful for producing non-natural hybrid seed from a seed-propagated crop plant, such as those described under the title "Making and Using Transgenic Plant Cells and Transgenic Plants", e.g., corn, rice, wheat, oats, barley, rye, triticale, millet, sorghum, quinoa, amaranth, buckwheat, fodder grass, peat, alfalfa, cotton, safflower, sunflower, soybeans, canola, rapeseed, flax, peanuts, beans, peas, lentils , alfalfa, lettuce, asparagus, artichokes, celery, carrots, radishes, cabbage, kale, mustard, broccoli, cauliflower, Brussels sprouts, turnips, kohlrabi, cucumbers, melons, strawberry squash, pumpkin, onions, leeks, shallots, chives, tomatoes, eggplant, pepper, physalis, beetroot, chard, spinach, ornamental plants, and forest trees. In a particularly preferred embodiment, the non-natural hybrid seed is corn seed. Non-natural hybrid seed produced using human activity that includes any of the embodiments of the method are specifically further claimed.

INDUCTIVELY STERILE TRANSGENIC PLANTS CONTAINING A RECOMBINANT DNA CONSTRUCTION

An independent aspect of this invention includes an inductively sterile transgenic plant that contains in its genome a recombinant DNA construct that transcribes RNA that includes: (a) at least one exogenous miRNA recognition site recognizable by a mature miRNA that is specifically expressed in tissue plant reproductive; and (b) messenger RNA encoding a protein that confers tolerance to a herbicide, wherein the mature miRNA specifically suppresses expression of the protein in reproductive tissue, and wherein sterility of the second parental plant is inducible by application of the herbicide to the plant. In one embodiment, the reproductive tissue is male reproductive tissue and the sterility is male sterility. In another embodiment, the reproductive tissue is female reproductive tissue and the sterility is female sterility. In a further embodiment, the reproductive tissue includes a fertilized egg, and sterility results in the failure of the fertilized egg to develop into a viable seed.

In preferred embodiments, the inductively sterile transgenic plant is a seed-propagated crop plant, e.g., maize, maize, rice, wheat, oats, barley, rye, triticale, millet, sorghum, quinoa, amaranth, buckwheat, grass forage, peat, alfalfa, cotton, safflower, sunflower, soybeans, canola, rapeseed, flax, peanuts, beans, peas, lentils, alfalfa, lettuce, asparagus, artichokes, celery, carrots, radishes, cabbage, kale, mustard, broccoli, cauliflower, Brussels sprouts, turnip, kohlrabi, cucumber, melons, pumpkin, pumpkin, onion, garlic, leek, shallots, chives, tomato, eggplant, pepper, physalis, beetroot, chard, spinach, ornamental plants, and forest trees.

Recombinant DNA Constructs: The inductively sterile transgenic plant provided by this invention has in its genome a recombinant DNA construct that transcribes RNA that includes: (a) at least one exogenous miRNA recognition site that transcribes RNA that includes: (a) at least one exogenous miRNA recognition site recognizable by a mature miRNA that is specifically expressed in plant reproductive tissue; and (b) messenger RNA encoding a protein that confers tolerance to a herbicide, wherein the mature miRNA specifically suppresses expression of the protein in reproductive tissue, and wherein the sterility of the second parental plant is inducible by application of the herbicide to the plant. Such a recombinant DNA construct is useful for making transgenic plant chromosomes, cells, plants and seeds, which contain such a construct, all of which are particularly useful for hybrid seed production.

In various embodiments of this invention, including embodiments of the inductively sterile transgenic plant, the recombinant DNA construct further includes at least one element selected from: (a) a plant promoter; (b) a gene suppression element; (c) an intron; (d) a gene expression element; (e) DNA that transcribes an RNA aptamer capable of binding a ligand (f) DNA that transcribes an RNA aptamer capable of binding a ligand and DNA that transcribes regulatory RNA capable of regulating the expression of a target gene, characterized in that regulation is dependent on the conformation of the regulatory RNA, and the conformation of the regulatory RNA is affected allosterically by the binding state of the ap-5 RNA tamer, and (g) at least one T-DNA border. These and other elements (e.g., terminators and spacer DNA) useful in this invention are described in detail below under the headings "Promoters11; "Gene Suppression Elements", "Introns", "Gene Expression Elements", "Aptamers" , "Binds", "Regulatory RNA", "T-DNA Borders", and 10 "Spacer DNA" and elsewhere in this description. Non-limiting examples of how these various elements may be arranged are described in Figures 1 and 2. Techniques for making and using recombinant DNA constructs of the invention, for making transgenic plant cells containing the recombinant DNA constructs and plants, seeds and plants of transgenic progeny derived from them, and to test the effects of transcribing the recombinant DNA constructs, are described in detail under the headings "Making and Using Recombinant DNA Constructs", "Making and Using Transgenic Plant Cells and Transgenic Plants " and elsewhere in this description. Promoters: Generally, the recombinant DNA construct includes a promoter operably linked to the DNA capable of being transcribed. Suitable promoters include any promoter that is capable of transcribing DNA in the plant cell where transcription is desired. Suitable non-constitutive promoters for use with the recombinant DNA constructs of the invention include spatially specific promoters, temporally specific promoters and inducible promoters. Spatially specific promoters may include organelle-, cell-, tissue-, or organ-specific promoters functional in a plant (e.g., a plastid-specific, root-specific, pollen-specific, or seed-specific promoter to suppress the expression of the first target RNA in plastids, roots, pollen, or seeds, respectively). In many cases, a seed-specific, embryo-specific, aleurone-specific, or endosperm-specific promoter is especially useful. Temporally specific promoters may include promoters that tend to promote expression during certain developmental stages in the growth or reproductive cycle of a plant, or during different times of day and night, or in different seasons in a year. Inducible promoters include promoters induced by chemicals (e.g., exogenous or synthetic chemicals as well as endogenous pheromones and other signaling molecules) or by environmental conditions such as, but not limited to, biotic or abiotic stress (e.g., lack of water or drought, heat, cold, high or low nutrient or salt levels, high or low light levels, or pest or pathogen infection). An expression-specific promoter may also include promoters that are generally expressed constitutively but in different degrees or "strengths" of expression, including promoters commonly thought of as "strong promoters" or as "weak promoters". Because the miRNA recognition site provides control of tissue-selective expression of the messenger RNA, 15 in many preferred embodiments the promoter is simply a constitutive promoter. .

Specific non-limiting examples of promoters useful in plants include an opaline synthase promoter isolated from Agrobacterium T-DNA, and a cauliflower mosaic virus 35S promoter, among others, as well as enhanced promoter elements or chimeric promoters, for example, an enhanced cauliflower mosaic virus (CaMV) 35S promoter linked to an enhancer element (an intron of the Zea mays heat shock protein 70). Many expression-specific promoters functional in plants and useful in aspects of the invention are known in the art. For example, U.S. Patents 5,837,848; 6,437,217 and 6,426,446 describe root-specific promoters; U.S. Patent 6,433,252 describes a corn L3 oleosin promoter; U.S. Patent Application Publication 2004/0216189 describes a promoter for a plant nuclear gene encoding a plastid-localized aldolase; U.S. Patent 6,084,089 describes cold-inducible promoters; U.S. Patent Number 6,140,078 describes salt-inducible promoters; U.S. Patent 6,294,714 describes light-inducible promoters; U.S. Patent 6,252,138 describes pathogen-inducible promoters; and U.S. Patent Application Publication 2004/0123347 A1 describes water-inducible promoters. The promoter element may include nucleic acid sequences that are non-naturally occurring promoters or promoter elements or homologues thereof, but which can regulate the expression of a gene. Examples of such "gene-independent" regulatory sequences include naturally occurring or artificially designed RNA sequences that include a ligand- or aptamer-binding region and a regulatory region (which may be cis-acting or trans-acting). example, Isaacs et al. (2004) Nat. Biotechnol., 22:841-847, Bayer & Smolke (2005) Nature Biotechno!., 23:337-343, Mandai & Breaker (2004) Nature Bev. ., 5:451-463, Davidson & Ellington (2005) Trends Biotechnol., 23:109-112, Winkler et al. (2002) Nature, 419:952-956, Sudarsan et al. 644-647, and Mandai & Breaker (2004) Nature Struct. Mol. Biol., 11:29-35. Such "riboregulators" could be selected or designed for specific spatial or temporal specificity, for example, to regulate gene translation. exogenous only in the presence (or absence) of a given concentration of the appropriate ligand.

Gene suppression elements: The gene suppression element may be DNA capable of being transcribed of any suitable length, and will generally include at least about 19 to about 27 nucleotides (e.g., 19, 20, 21, 22, 23, or 24 nucleotides) for each target gene that the recombinant DNA construct is designed to suppress. In many embodiments the gene suppression element includes more than 23 nucleotides (e.g., more than about 30, about 50, about 100, about 200, about 300, about 500, about 1000, about 1500, about 2000, about 3000, about 4000, or about 5000 nucleotides) for each target gene that the recombinant DNA is designed to suppress. Target genes are described under the heading "Target Genes".

Suitable gene suppression elements useful in the recombinant DNA constructs of the invention include at least one element (and, in some embodiments, multiple elements) selected from the group consisting of: (a) DNA that includes at least DNA segment antisense that is antisense to at least one segment of at least one first target gene; (b) DNA that includes multiple copies of at least one segment of antisense DNA that is antisense to at least one segment of at least one first target gene; (c) DNA that includes at least one segment of sense DNA that is at least one segment of at least one first target gene; (d) DNA that includes multiple copies of at least one sense DNA segment that is at least one segment of at least one first target gene; (| (e) DNA that transcribes RNA to suppress at least one first target gene by forming double-stranded RNA and includes at least one antisense DNA segment that is antisense to at least one segment of the at least one first target gene; (f) DNA that transcribes RNA to suppress at least one first target gene by forming a double-stranded RNA and includes multiple serial segments of antisense DNA that are antisense to at least one segment of the at least one. a first target gene and multiple serial segments of sense DNA that are at least one segment of the at least one first target gene; (g) DNA that transcribes RNA to suppress at least one first target gene by forming multiple double strands. of RNA and includes multiple segments of antisense DNA that are antisense to at least one segment of the at least one first target gene and multiple segments of sense DNA that are at least one segment of the at least one first target gene, and wherein said multiple antisense DNA segments and multiple sense DNA segments are arranged in a series of inverted repeats; (h) DNA that includes nucleotides derived from a miRNA, preferably a plant miRNA; (i) DNA that includes nucleotides from an siRNA; (j) DNA that transcribes an RNA aptamer capable of binding a ligand; and (k) DNA that transcribes an RNA aptamer capable of binding a ligand, and DNA that transcribes regulatory RNA capable of regulating the expression of at least one first target gene, wherein the regulation is dependent on the conformation of the RNA regulatory, and the conformation of regulatory RNA is allosterically affected by the binding state of the RNA aptamer.

Non-limiting representations of these gene suppression elements are illustrated in Figure 2. Any of these gene suppression elements, transcribing to a single double-stranded RNA or to multiple double-stranded RNAs, can be designed to suppress more than one target gene, including , for example, more than one allele of a target gene, multiple target genes (or multiple segments of at least one target gene) from a single species, or target genes from different species. Aptamers, regulatory RNA, and ligands are described under their respective headings below.

Antisense DNA Segments: In one embodiment, the at least one antisense DNA segment that is antisense to at least one segment of the at least one first target gene includes DNA sequence that is antisense or complementary to at least one segment of the at least one first target gene, and may include multiple antisense DNA segments, that is, multiple copies of at least one antisense DNA segment that is antisense to at least one segment of the at least one target gene. least one first target gene. Multiple segments of antisense DNA may include DNA sequence that is antisense or complementary to multiple segments of the at least one first target gene, or to multiple copies of a segment of the at least one first target gene, or to segments of multiple first target genes, or any combination thereof. Multiple antisense DNA segments can be fused into a chimera, for example, which includes DNA sequences that are antisense to multiple segments of one or more first target genes and fused together.

The antisense DNA sequence that is antisense or complementary to (i.e., can form Watson Crick base pairs with) at least one segment of the at least one first target gene is preferably at least about 80 %, or at least about 85%, or at least about 90%, or at least about 95% complementarity to at least one segment of the at least one first target gene. In a preferred embodiment, the DNA sequence that is antisense or complementary to at least one segment of the at least one first target gene has between about 95% to about 100% complementarity to at least one segment of the at least one first target gene. a first target gene. Where the at least one antisense DNA segment includes multiple antisense DNA segments, the degree of complementarity may be, but need not be, identical for all multiple antisense DNA segments. Sense DNA segments: In another embodiment, the at least one sense DNA segment that is at least a segment of the at least one first target gene includes DNA sequence that corresponds to (i.e., has a sequence that is identical or substantially identical a) at least one segment of the at least one first target gene, and may include multiple segments of sense DNA, that is, multiple copies of at least one segment of sense DNA that corresponds to (that is, has the nucleotide sequence of ) at least one segment of the at least one first target gene. Multiple segments of sense DNA may include DNA sequence 20 that is or corresponds to multiple segments of the at least one first target gene, or to multiple copies of a segment of the at least one first target gene, or to segments of multiple target genes, or any combination of these. Multiple segments of sense DNA can be fused into a chimera, that is, they can include DNA sequences that correspond to multiple segments of one or more first target genes and fused together.

The sense DNA sequence corresponding to at least one segment of the target gene preferably has at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identity. sequence to at least one segment of the target gene. In a preferred embodiment, the DNA sequence corresponding to at least one segment of the target gene has between about 95% to about 100% sequence identity to at least one segment of the target gene. Where the at least one sense DNA segment includes multiple sense DNA segments, the degree of sequence identity may, but need not, be identical for all multiple sense DNA segments.

Multiple copies: where the gene suppression element includes 5 multiple copies of antisense DNA sequence or multiple copies of sense DNA sequence, these multiple copies can be arranged serially in tandem repeats. In some embodiments, these multiple copies can be arranged serially end-to-end, that is, in directly connected tandem repeats. In some embodiments, these multiple copies may be arranged in interrupted tandem repeats, where one or more segments of spacer DNA may be located adjacent to one or more of the multiple copies. Tandem repeats, directly linked or interrupted, or any combination of both, may include multiple copies of a single antisense DNA sequence or multiple copies of a single sense DNA sequence in a serial arrangement or may include multiple copies of more of an antisense DNA sequence or more than one sense DNA sequence in a serial arrangement. Double-stranded RNA: in those embodiments wherein the gene suppression element includes at least one antisense DNA segment that is antisense to at least one segment of the at least one target gene or at least one sense DNA segment that is at least At least one segment of the at least one target gene, RNA transcribed from any of at least one antisense DNA or at least one sense DNA may become double stranded by the action of an RNA-dependent RNA polymerase. See, for example, U.S. Patent number 5,283,184.

In still other embodiments, the gene suppression element may include DNA that transcribes RNA to suppress the at least one first target gene by forming double-stranded RNA and includes at least one antisense DNA segment that is antisense to at least one first target gene. at least one segment of the at least one target gene (as described above under the heading "Antisense DNA Segments") and at least one segment of sense DNA that is at least one segment of the at least one first target gene (as described above). described above under the heading "Sense1 DNA Segments'). Such a gene suppression element may additionally include spacer DNA segments. Each at least one antisense DNA segment is complementary to at least a portion of a sense DNA segment to in order to allow the formation of double-stranded RNA by intramolecular hybridization of the at least one antisense DNA segment and the at least one sense DNA segment. Such complementarity between a sense DNA segment and an antisense DNA segment. meaning can, but does not need to be, 100% complementarity; In some embodiments, such complementarity 10 may preferably be at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% complementarity. Double-stranded RNA can be in the form of a single dsRNA stem (base pairing region between sense and antisense strands), 15 or it can have multiple dsRNA stems. In one embodiment, the gene suppression element may include DNA that transcribes RNA to suppress the at least one first target gene by essentially forming a single double-stranded RNA and includes multiple serial antisense DNA segments that are antisense to at least a segment of the at least one first target gene and multiple serial sense DNA segments that are at least one segment of the at least one first target gene; the multiple serial antisense segments and multiple serial sense segments can form a single stem or multiple double-stranded RNA stems in a serial arrangement (with or without a 25-base unpaired DNA spacer separating the multiple stems) . In another embodiment, the gene suppression element includes DNA that transcribes RNA to suppress the at least one first target gene by forming multiple dsRNA RNA stems and includes multiple antisense DNA segments that are antisense to at least a segment of the at least one first target gene and multiple segments of sense DNA which are at least one segment of the at least one first target gene, and wherein said multiple segments of antisense DNA and the multiple segments of DNA sense are arranged in a series of dsRNA "stems" (such as, but not limited to, "inverted repeats"). Such multiple dsRNA "stems" can further be arranged in arrays or groups to form inverted tandem repeats, or structures resembling "hammerhead" or "cloverleaf" shapes. Any of these gene suppression elements may further include spacer DNA segments found within a dsRNA stem (e.g., as a spacer between multiple antisense and sense DNA segments or as a spacer between an antisense DNA segment). -sense and a base-paired segment of sense DNA) or outside a double-stranded RNA stem (e.g., as a loop region separating a pair of inverted repeats). In cases where base-paired antisense and sense DNA segments are of unequal length, the longer segment can act as a spacer. Figure 2 represents illustrations of possible embodiments of these gene suppression constructs. miRNAs: In a further embodiment, the gene suppression element may include DNA that includes nucleotides derived from a miRNA (microRNA), that is, a DNA sequence that corresponds to a native miRNA to a virus or eukaryote of interest (including plants and animals, especially invertebrates), or a DNA sequence derived from such native miRNA but modified to include nucleotide sequences that do not correspond to the native miRNA. Although miRNAs have not been reported to date in fungi, fungal miRNAs, which should exist, are also suitable for use in the invention. A preferred embodiment includes a gene suppression element containing DNA that includes nucleotides derived from a viral or plant miRNA. In a non-limiting example, nucleotides derived from a miRNA may include DNA that includes nucleotides that correspond to the loop region of a native miRNA, and nucleotides derived from a miRNA may include DNA derived from a miRNA precursor sequence, such as primRNA sequence. or native pre-miRNA, or nucleotides that correspond to regions of a native miRNA and nucleotides that are selected from a target gene sequence number such that the overall structure (e.g., the placement of mismatches in the base structure of the pre -miRNA) is preserved to allow the pre-miRNA to be processed into a mature miRNA. In yet another embodiment, the gene suppression element may include DNA that includes nucleotides derived from a miRNA and is capable of inducing or guiding in-phase cleavage of an endogenous transcript into transacting siRNAs, as described by Allen et al. (2005) Cell, 121:207-221. Thus, DNA that includes nucleotides derived from a miRNA may include naturally occurring sequence in a miRNA or a miRNA precursor molecule, synthetic sequence, or both. siRNAs: in yet another embodiment, the gene suppression element may include DNA that includes nucleotides of a small interfering RNA (siRNA). The siRNA may be one or more native siRNAs (such as siRNAs isolated from a non-transgenic eukaryote or a transgenic eukaryote), or it may be one or more DNA sequences predicted to have siRNA activity (such as by use of known predictive tools). - 15 of the art, see, for example, Reynolds et al. (2004) Nature Biotechnol., 22:326-330). Multiple native or predicted siRNA sequences can be joined into a chimeric siRNA sequence for gene suppression. Such DNA that includes nucleotides from an siRNA preferably includes at least 19 nucleotides, and in some embodiments preferably includes at least 20, at least 22, at least 23, or at least 24 nucleotides. In other embodiments, the DNA that includes nucleotides from an siRNA may contain substantially more than 21 nucleotides, e.g., more than about 50, about 100, about 300, about 500, about 1000, about 3000 , or about 5000 nucleotides or more.

Target genes: The gene suppression element can be designed to suppress any first target gene. In some embodiments, the construct further includes a second gene suppression element to suppress at least one second target gene, wherein the second gene suppression element is located adjacent to the intron. Whether a first or a second target gene, the target gene may include a single gene or part of a single gene that is targeted for suppression or may include, for example, multiple consecutive segments of a target gene, multiple non-consecutive segments of a target gene, multiple alleles of a target gene, or multiple target genes from one or more species. The target gene may be translatable (coding) sequence, or may be non-coding sequence (such as non-coding regulatory sequence), or both, and may include at least one gene selected from the group consisting of a eukaryotic target gene, a non-eukaryotic target gene, a microRNA precursor DNA sequence, and a microRNA promoter. The target gene may be native (endogenous) to the cell, (for example, a plant or animal cell) in which the recombinant DNA construct of the invention is transcribed. The target gene may be an exogenous gene, such as a transgene in a plant. A target gene may be a native gene targeted for suppression, with or without concomitant expression of an exogenous transgene, for example, by inclusion of a gene expression element in the same recombinant DNA construct or in a separate recombinant DNA construct. For example, it may be desirable to replace a native gene with a homologous exogenous transgene. The target gene may include a single gene or part of a single gene that is targeted for suppression, or may include, for example, multiple consecutive segments of a target gene, multiple non-consecutive segments of a target gene, multiple alleles of a gene target, or multiple target genes from one or more species. A target gene sequence may include any sequence from any species (including, but not limited to, non-eukaryotes such as bacteria, and viruses; fungi, plants; including monocots and dicots, such as crop plants, ornamental plants, and non-domesticated or wild; invertebrates such as arthropods, annelids, nematodes, and molluscs; and vertebrates such as amphibians, fish, birds, domestic or wild mammals, or even humans. The target gene is exogenous to the plant in which the construct is to be transcribed, but endogenous to a pest or pathogen (e.g., viruses, bacteria, fungi, oomycetes, and invertebrates such as insects, nematodes, and molluscs) of the plant. target may include multiple target genes, or multiple segments of one or more genes. In a preferred embodiment, the target gene or genes are a gene or genes from a pest or invertebrate plant pathogen. Such recombinant DNA constructs are particularly useful in providing transgenic plants that have resistance to one or more plant pests or pathogens, for example, resistance to a nematode such as a soybean cyst nematode or root knot nematode or to a harmful insect. The target gene may be a translatable (coding) sequence, or it may be a non-coding sequence (such as a non-coding regulatory sequence), or both. Non-limiting examples of a target gene include non-translatable (non-coding) sequences, such as, but not limited to, 5' untranslated regions, promoters, enhancers, or other non-coding transcriptional regions, 3' untranslated regions , terminators and introns. Target genes include genes encoding microRNAs, small interfering RNAs, RNA components of ribosomes or ribozymes, 15 small nucleolar RNAs, and other non-coding RNAs (see, for example, publicly provided non-coding RNA sequences at rfam.wustl .edu; Erdmann et al. (2001) Nucleic Acids Res., 29:189-193; Gottesman (2005) Trends Genet, 21:399-404; 33:121-124). A specific example of a target gene includes a microRNA recognition site (i.e., the site on an RNA strand to which mature miRNA binds and induces cleavage). Another specific example of a target gene includes a microRNA precursor sequence. native to a transgenic plant pest or pathogen, that is, the primary transcript encoding a microRNA, or the RNA intermediates processed from that primary transcript (e.g., a primRNA or nuclear-limited pre-miRNA that can be exported from the nucleus to the cytoplasm). See, for example, Lee et al. (2002) EMBO Journal, 21:4663-4670; Reinhart et al. (2002) Genes & Dev., 16:161611626; Lund et al. (2004) Science, 303:95-98; and Millar & Waterhouse (2005) Fund. Integra Genomics, 5:129-135. Target genes may also include translatable (coding) sequence for genes encoding transcription factors and genes encoding enzymes involved in the biosynthesis or catabolism of molecules of interest (such as, but not limited to, amino acids, fatty acids, and others). lipids, sugars, and other carbohydrates, biological polymers, and secondary metabolites including alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin).

In many preferred embodiments, the target gene is an essential gene of the pest or plant pathogen. Essential genes include genes that are necessary for the development of the pest or pathogen into a fertile reproductive adult. Essential genes include genes that, when silenced or suppressed, result in the death of the organism (as an adult or at any stage of development, including gametes) or in the inability of the organism to reproduce successfully (e.g., sterility in a relative male or female or lethality to the zygote, embryo or larva). A description of essential nematode genes is found, for example, in Kemphues K. "Essential Genes" (24 December 2005), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/15 wormbook.1.57.1, available online at www.wormbook.org. Non-limiting examples of essential nematode genes include major sperm protein, RNA polymerase II, and chitin synthase (see, for example, U.S. Patent Application Publication US20040098761 A1); Additional essential soybean cyst nematode genes are provided in U.S. Patent Application 11/360,355, filed February 23, 2006. A description of insect genes is publicly available in the Drosophila genome database (available online). at flybase.bio.indiana.edu/). The majority of predicted Drosophila genes were analyzed for function by a cell culture-based RNA interference screen, resulting in 438 essential genes being identified; see, Boutros et al. (2004) Science, 303:832-835, and supporting material available online at www.science-mag.org/cgi/content/full/303/5659/832/DC1. A description of essential bacterial or fungal genes is provided in the Database of Essential Genes ("DEG", available online at tubic.tju.edu.cn/deg/); see Zhang et al. (2004) Nucleic Acids Res., 32:D271-D272.

Harmful plant invertebrates include, but are not limited to, harmful nematodes, harmful molluscs (slugs and snails), and harmful insects. Plant pathogens of interest include fungi, oomycetes, bacteria (e.g., the bacteria that cause leaf spots, fireblight, crown gall, and bacterial wilt), mollicutes, and viruses (e.g., the viruses that cause mosaics). , vein banding, stains, stains, or abnormal growth). See also G. N. Agrios, "Plant Pathology" (Fourth Edition), Academic Press, San Diego, 1997, 635 pp., for descriptions of fungi, bacteria, mollicutes (including mycoplasmas and spiroplasmas), viruses, nematodes, higher parasitic plants, and flagellated protozoa, all of which are plant pests or pathogens of concern. See also the continually updated compilation of plant pests and pathogens and the diseases caused by them in "Common Names of Plant Diseases" of the American Phytopathological Society compiled by the Committee on Standardization of Common Names for Plant Diseases of the American Phytopathological Society. 1978-2005, available online at www.apsnet.orq/online/common/top.asp.

Non-limiting examples of fungal plant pathogens of particular interest include, for example, fungi that cause powdery mildew, rust, leaf blight, damping-off, root rot, crown rot, cotton boll rot, stem canker, twig blight, vascular wilt, soot, mold, including, but not limited to, Fusaríum spp., Phakospora spp., Rhizoctonia spp., Aspergillus spp., Gibberella spp., Pyrícularía spp., and Alternaria spp.. Specific examples of Fungal plant pathogens include Phakospora pachirhizi (Asian soybean rust), Puccinia sorghi (common corn rust), Puccinia polysora (Southern corn rust), Fusarium oxysporum and others Fusarium spp., Alternaria spp., Penicillium spp., Rhizoctonia solani, Exserohilum turcicum (Northern corn leaf spot), Bipolaris maydis (Southern corn leaf spot), Ustilago maydis (corn sooty mold), Fusaríum graminearum (Gibberella zeae), Fusaríum verticilliodes (Gibberella moniliformis), F. proliferatum ( G. fujikuroi var. intermedia), F. subglutinans (G. subglutinans), Diplodia maydis, Sporisoríum holci-sorghi, Colletotrichum graminicola, Setosphaería turcica, Aureobasidi-a zeae, Sclerotinia sclerotíorum, and the various fungal species provided in Tables 4 and 5 of U.S. Patent 6,194 .636. Non-limiting examples of plant pathogens include pathogens previously classified as fungi but more recently classified as oomycetes. Specific examples of plant pathogenic oomycetes of particular interest include members of the genera Pythium (e.g., Pythium aphanidermatum) and Phytophthora (e.g., Phytophthora infestans, Phytophthora soyae,) and organisms that cause downy mildew (e.g., Peronospora farinosa).

Non-limiting examples of bacterial pathogens include mycoplasmas that cause yellowing and spiroplasmas such as Spiroplasma kunkelii, which causes corn stunting, eubacteria such as Pseudomonas avenae, Pseudomonas andropogonis, Erwinia stewartii, Pseudomonas syringae pv. syringae, Xylella fastidiosa, and the various bacterial species listed in Table 3 of U.S. Patent 6,194,636.

Non-limiting examples of viral plant pathogens of particular interest include maize dwarf mosaic virus (MDMV), sugar cane mosaic virus (SCMV, formerly MDMV strain B), wheat streak mosaic virus (Wheat streak mosaic virus, WSMV), maize chlorotic dwarf virus (MCDV), barley yellow dwarf virus (BYDV), banana bunch disease virus (BBTV), and the various viruses listed in Table 2 of U.S. Patent 6,194,636. Invertebrate pests of particular interest, especially in, but not limited to, regions of the Southern Hemisphere (including South and Central America) including aphids, rootworm, spodoptera, noctuidae, potato beetle, Lygus spp., any hemiptera, or homoptera , or heteropteran, any lepidopteran, any coleopteran, nematodes, moth caterpillars, earworms, fall armyworms, borers, leafroller caterpillars, and others. Arthropod pests specifically encompassed by this invention include various species of caterpillars including caterpillar (Agrotis replete), threadworm (Agrotis ipsilorí), caterpillar (Anicla ignicans), granulated caterpillar (Feltia subterranean), 'roughworm' (Agrotis malefida)', Mediterranean flour moth (A-nagasta kuehniella), square-necked grain beetle (Cathartus quadricollis), jumping flea beetle (Chaetocnema spp), moth (Corcyra cephalonica), corn rootworm or San Antonio" (Diabrotica speciosa), sugarcane borer (Diatraea saccharalis), collar borer (Elasmopalpus lignosellus), brown stink bug (Euschistus spp.), corn earworm (Helicoverpa zeá), grain beetle ( Laemophloeus minutus), grass leafworm (Moeis latipes), beetle (Oryzaephilus surinamensis), flour moth (Pyralis farinalis), Indian meal moth (Plodia interpunctella), corn aphid (Rhopalosiphum maidis), brown stink bug or "subterranean chinch" (Scaptocoris castaneà), green aphid (Schizaphis graminum), cereal weevil (Sitophilus zeamais), cereal tinea (Sitotroga cerealella), fall armyworm (Spodoptera frugíperda), beetle (cadelle beetle) (Tenebroides maurítanicus), two-spotted spider mite (Tetranychus urticae), brown flour beetle (Triboleum castaneum), cotton leafworm (Alabama argillacea), cotton weevil (Anthonomus grandis), cotton aphid (Aphis gossypii), potato whitefly bollworm (Bemisia tabaci), several species of thrips (Frankliniella spp.), bollworm (Helicoverpa zea), "oruga bolillera" (e.g. Helicoverpa geletopoeorí), apple bollworm (Heliothis virescens), green stink bug (Neza- ra viridula), pink armyworm (Pectinophora gossypiella), fall armyworm (Spodoptera exigua), mites (Tetranychusspp.), onion thrips (Thrips ta-~bad), greenhouse whitefly (Trialeurodes vaporarium), defoliant caterpillar ( Anticarsia gemmatalis), corn hatchet beetle or "astylus moteado" (Astylus atromaculatus), "alfalfa oruga" (Colias lesbia), "brown chinche" or "chinche de los cuernos" (Dichelops furcatus), "alquiche chico " (Edessa miditabunda), blister beetles (Epicauta spp.), "barrenador dei brote" (Epinotia aporema), "oruga verde dei yuyo colorado" (Loxostege bifidalis), root-knot nematodes (Meloidogyne spp.) , "oruga cuarteadora" (Mods repanda), southern green stink bug (Nezara viridula), "chinche de la alfalfa" (Píezodorus guildinii), green cloverworm (Plathypena scabra), soybean caterpillar (Pseudoplusia includens), caterpillar "isoca metera del girasof' (Rachiplusia nü), "yellow woolybeaí" (Spilosoma virginica), yellow striped armyworm (Spodoptera ornithogalli), various root weevils (family Curculionidae), various "wireworms" (family Elateridae), and various corós (family Scarabaeidae). Nematode pests specifically encompassed by this invention include pests of corn (Belonolaimus spp., Tríchodorus spp., Longidorus spp., Dolichodorus spp., Anguina spp., Pratylenchus spp., Meloidogyne spp., Heterodera spp.), soybean (Heterodera glycines, Meloidogyne spp., Belonolaimus spp.), bananas {Radopholus similis, Meloidogyne spp., Helicotylenchus spp.), sugar cane {Heterodera sacchari, Pratylenchus spp., Meloidogyne spp.), oranges {Tylenchulus spp., Radopholus spp., Belonolaimus spp., Pratylenchus spp., Xiphinema spp.), coffee {Meloidogyne spp., Pratylenchus spp.), coconut palm {Bursaphelenchus spp.), tomatoes {Meloidogyne spp., Belonolaimus spp., Nacobbus spp.), grapes {Meloidogyne spp. , Xiphinema spp., Tylenchulus spp., Criconemella spp.), lemon and lime {Tylenchulus spp., Radopholus spp., Belonolaimus spp., Pratylenchus spp., Xiphinema spp.), cocoa {Meloidogyne spp., Rotylenchulus reniformis), pineapple {Meloidogyne spp., Pratylenchus spp., Rotylenchulus reniformis), papaya {Meloidogyne spp., Rotylenchulus reniformis), grapefruit {Tylenchulus spp., Radopholus spp. Belonolaimus spp., Pratylenchus spp., Xiphinema spp., and broad beans {Me-loidogyne spp.).

Pest target genes may include invertebrate genes for sperm major protein, alpha tubulin, beta tubulin, vacuolar ATPase, glyceraldehyde-3-phosphate dehydrogenase, RNA polymerase li, chitin synthase, cytochromes, miRNAs, miRNA precursor molecules, miRNA promoters , as well as other genes such as those described in U.S. Patent Application Publication 2006/0021087 A1, PCT Patent Application PCT/US05/11816, and in Table II of U.S. Patent Application Publication 2004/0098761 A1. Pathogen target genes may include genes for viral translation initiation factors, viral replicases, miRNAs, miRNA precursor molecules, fungal tubulin, fungal vacuolar ATPase, fungal chitin synthase, fungal MAP kinases, fungal Pad Tyr/Thr phosphatase, enzymes involved in nutrient transport (e.g., amino acid transporters or sugar transporters), enzymes involved in fungal cell wall biosynthesis, cutinases, melanin, biosynthetic enzymes, polygalacturonases, pectinases, pectin lyases, cellulases, proteases, genes that interact with plant avirulence genes, and other genes involved in pathogen invasion and replication in the infected plant. Thus, a target gene does not need to be endogenous to the plant in which the recombinant DNA construct is transcribed. A recombinant DNA construct of the invention can be transcribed in a plant and used to suppress a gene of a pathogen or pest that may infest the plant.

Specific non-limiting examples of suitable target genes also include amino acid catabolic genes (such as, but not limited to, the maize LKF1/SDH gene encoding lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), and their homologues ), corn zein genes, genes involved in fatty acid synthesis (e.g., microsomal fatty acid desaturases and plant acyl-ACP thioesterases, such as, but not limited to, those described in U.S. Patent Nos. 6,426,448, 6,372,965, and 6,872,872), genes involved in multi-step biosynthesis pathways, where it may be of interest to regulate the level of one or more intermediates, such as genes encoding enzymes for polyhydroxyalkanoate biosynthesis (see, for example, example, U.S. Patent No. 5,750,848); and genes encoding cell cycle control proteins, such as proteins with cyclin-dependent kinase (CDK) inhibitor-like activity (see, for example, genes described in International Patent Application Publication number WO 05007829A2). Target genes may include genes encoding undesirable proteins (e.g., allergens or toxins) or enzymes for the biosynthesis of undesirable compounds (e.g., undesirable taste or odor components). Thus, one embodiment of the invention is a transgenic plant or tissue from such a plant that is improved by the suppression of allergenic proteins or toxins, for example, a peanut, soybean, wheat seed with decreased allergenicity. Target genes may include genes involved in fruit ripening, such as polygalacturonase. Target genes may include genes where expression is preferably limited to a particular cell or tissue or developmental stage, or where expression is preferably transient, that is, where constitutive or general suppression, or suppression that spreads across many tissues. , is not necessarily desired. Thus, other examples of suitable target genes include genes that encode proteins that, when expressed in transgenic plants, make the transgenic plants resistant to pests or pathogens (see, for example, genes for cholesterol oxidase as described in U.S. Patent No. 5,763. 245); genes where expression is induced by the pest or pathogen; and genes that can induce or restore fertility (see, for example, barstar/barnase genes described in U.S. Patent No. 6,759,575). The recombinant DNA constructs of the invention can be designed to more specifically suppress the target gene by designing the gene suppression element or elements to include regions substantially non-identical to a non-target gene sequence. Non-target genes may include any gene that is not intended to be silenced or suppressed, both in a plant that transcribes the recombinant DNA construct and in organisms that may come into contact with RNA transcribed from the recombinant DNA construct. A non-target gene sequence may include any sequence from any species (including, but not limited to, non-eukaryotes such as bacteria, and viruses; fungi, plants, including monocots and dicots, such as crop plants, ornamentals, and wild or non-domesticated plants; invertebrates such as arthropods, annelids, nematodes, and mollusks; and vertebrates such as amphibians, fish, birds, domestic or wild mammals, and even humans).

In one embodiment, the target gene is an endogenous gene for a given species, such as a given plant (such as, but not limited to, agriculturally or commercially important plants, including monocots and dicots), and the non-target gene it may be, for example, a gene from a non-target species, such as another plant species or a gene from a virus, fungus, bacteria, invertebrate, or vertebrate, even a human. A non-limiting example is where the gene suppression element is designed to suppress a target gene that is an endogenous gene for a single species (e.g., Western corn rootworm, Diabrotica virgifera virgifera LeConte) but does not suppress a non- target such as the genes of related, even closely related, species (e.g., Northern corn rootworm, Diabrotica barberí Smith & Lawrence, or Southern corn rootworm, Diabrotica undecimpunctata).

In other embodiments (e.g., where it is desirable to suppress a target gene across multiple species), it may be desirable to design the gene suppression element to suppress a target gene sequence common to the multiple species in which the target gene is to be silenced. In this way, a gene suppression element can be selected to be specific to one taxon (e.g., specific to a genus, family, or even a larger taxon such as a phylum, e.g., arthropods), but not to other taxa. (e.g. plants or vertebrates or mammals). In a non-limiting example of this embodiment, a gene suppression element for gene silencing can be selected to target pathogenic fungi (e.g., a Fusarium spp.), but not target any other beneficial fungal gene sequence.

In another non-limiting example of this embodiment, a gene suppression element for gene silencing in corn rootworm can be selected to be specific for all members of the hl genus.

Diabrotica. In a further example of this embodiment, such a gene suppression element targeting Diabrotica may be selected so as not to target any gene sequences of beneficial coleopterans (e.g., predatory coccinellid beetles, commonly known as ladybugs) or other species of beneficial insects. The necessary degree of specificity of an RNA to silence a target gene depends on several factors. For example, the RNA to silence a target gene includes double-stranded RNA (dsRNA), and so factors may include the size of the smallest dsRNA fragments that are expected to be produced by the action of Dicer or Dicer-like proteins, and the relative importance of decreasing the potential of dsRNAs to suppress non-target genes. For example, where dsRNA fragments are expected to be 21 base pairs in size, a particularly preferred embodiment includes RNA to silence a target gene that encodes regions substantially non-identical to a non-target gene sequence, such as regions within which each contiguous fragment that includes at least 21 nucleotide pairings of less than 21 (e.g., of less than 21, or of 30 less than 20, or of less than 19, or of less than 18 , or less than 17) of the 21 contiguous nucleotides of a non-target gene sequence. In another embodiment, regions substantially non-identical to a non-target gene sequence include regions within which each contiguous fragment that includes at least 19 nucleotide pairings less than 19 (e.g., 19, or less than 18, or less than 17, or less than 16) of the 19 contiguous nucleotides of a non-target gene sequence.

In some embodiments, it may be desirable to design the RNA for silencing a target gene to include regions predicted not to generate undesirable polypeptides, for example, by screening the RNA for silencing a target gene for sequences that may encode known undesirable polypeptides or close homologs. of these. Undesirable polypeptides include, but are not limited to, polypeptides homologous to known allergenic polypeptides and polypeptides homologous to known polypeptide toxins. Publicly available sequences encoding such potentially allergenic peptides are available, for example, in the Food Allergy Research and Resource Program (FARRP) allergen database (available at allergenonline.com) or the Biotechnology Information for Food Safety Databases (available at www.iit.edu/~sgendel/fa.htm) (see also, for example, Gendel (1998) Adv. Food Nutr. Res., 42:63-92). Undesirable sequences may also include, for example, those polypeptide sequences identified as known toxins or potential or known allergens and contained in publicly available databases such as GenBank, EMBL, SwissProt, and others, which are searchable by the Entrez system (www .ncbi.nih.gov/Entrez). Non-limiting examples of potentially allergenic peptide sequences include soy glycinin, peanut oleosin and agglutinin, wheat glutenins, casein, lactalbumin, and lactoglobulin from bovine milk and tropomyosin from various shellfish (allergenonline.com). Non-limiting examples of undesirable potentially toxic peptides include tetanus toxin tetA from Clostridium tetani, diarrheal toxins from Staphylococcus aureus, and venoms such as conotoxins from Conus spp. and neurotoxins from arthropods and reptiles (www.ncbi.nih.gov/Entrez).

In a non-limiting example, an RNA for silencing a target gene is screened to eliminate those sequences capable of being translated that encode polypeptides with perfect homology to a known allergen or toxin for 8 contiguous amino acids, or with at least 35% identity per at least minus 80 amino acids; such scans can be performed on any and all possible reading frames in both directions, on potential open reading frames starting with AUG (ATG in the corresponding DNA), or on all possible open reading frames, regardless of whether they start with an AUG (or ATG) or not. When a "hit" or pairing is made, that is, when a sequence encoding a potential polypeptide with perfect homology to a known allergen or toxin for 8 consecutive amino acids (or at least about 35% identity per at least about 80 amino acids), is identified, the nucleic acid sequences that match the hit can be avoided, eliminated, or modified when selecting sequences to be used in an RNA to silence a target gene.

Avoiding, eliminating, or modifying an unwanted sequence can be achieved by any of a number of methods known to those skilled in the art. In some cases, the result may be new sequences that are believed not to exist naturally. For example, avoidance of certain sequences can be achieved by splicing "clean" sequences into new chimeric sequences to be used in an RNA to silence a target gene. Since RNA for silencing a target gene includes double-stranded RNA (dsRNA), we recognize that in some dsRNA-mediated silencing, it is possible that dsRNA sequences that do not pair perfectly are effective in gene silencing. For example, it has been shown that mismatches near the center of a miRNA complementary site have stronger effects on miRNA gene silencing than pairings located more distally. See, for example, Figure 4 in Mallory et al. (2004) EMBO J., 23:3356-3364. In another example, it was reported that both the position of an unpaired base pair and the identity of the nucleotides forming the unpaired influence the ability of a given siRNA to silence a target gene, and that non-adenine-cytosine pairings, in addition to the G:U wobble base pair, they were well tolerated (see Du et al. (2005) Nucleic Acids Res., 33:1671-1677). Thus, the dsRNA that is included in the RNA to silence a target gene need not always have 100% sequence identity with the intended target gene, but generally would preferably have substantial sequence identity with the intended target gene, such as about 95 %, about 90%, about 85%, or about 80% sequence identity with the intended target gene. One skilled in the art would be able to judge the importance given to screening for regions predicted to be more highly specific to the target gene or predicted not to generate undesirable polypeptides, relative to the importance given to other criteria, such as, but not limited to, percentage of sequence identity with the intended target gene or the predicted gene silencing efficiency of a given sequence. For example, it may be desirable for an RNA for silencing a target gene to be active across multiple species, and therefore one of skill in the art may determine that it is more important to include in the RNA for silencing a target gene regions specific to the various species of interest, but less important to screen for regions predicted to have greater gene silencing efficiency or for regions predicted to generate undesirable polypeptides.

RNA aptamers: Nucleic acid aptamers are nucleic acid molecules that bind to a ligand through a binding mechanism that is not primarily based on Watson-Crick base pairing (as opposed to, for example, base pairing that occurs between complementary, anti-parallel nucleic acid strands to form a double-stranded nucleic acid structure). See, for example, Ellington & Szostak (1990) Nature, 346:818-822. A nucleic acid aptamer generally includes a primary nucleotide sequence that allows the aptamer to form a secondary structure (e.g., by forming "stem-loop" structures) that allows the aptamer to bind its ligand. The binding of the aptamer to its ligand is preferably specific, allowing the aptamer to distinguish between two or more molecules that are structurally similar (see, for example, Bayer & Smolke (2005) Nature Biotechnol., 23:337-343). Aptamers useful in the invention may, however, be monovalent (binding to a single ligand) or multivalent (binding to more than one individual ligand, for example, binding to a unit of two or more different ligands). See, for example, Di Giusto & King (2004) J. Biol. Chem., 279:46483-46489, which describes the design and construction of multivalent, circular DNA aptamers.

Aptamers useful in the invention may include DNA, RNA, nucleic acid analogs (e.g., peptide nucleic acids), blocked nucleic acids, chemically modified nucleic acids, or combinations thereof. See, for example, Schmidt et al. (2004) Nucleic Acids Fies., 32:5757-5765, which describe blocked nucleic acid aptamers. In a preferred embodiment of the invention, the aptamer is an RNA aptamer. In a particularly preferred embodiment, the aptamer is produced by in planta transcription. Examples of aptamers can be found, for example, in the public Aptamer Database, available online at aptamer.icmb.utexas.edu (Lee et al. (2004) Nucleic Acids Res., 32(1):D95-100 ).

Aptamers can be designed for a given ligand by various procedures known in the art, including in vitro selection or directed evolution techniques. See, for example, "SELEX" ("systematic evolution of ligands by exponential enrichment"), as described in Tuerk & Gold (1990) Science, 249:505-510, Ellington & Szostak (1990) Nature, 346:818- 822, Ellington & Szostak (1992) Nature, 355:850-852, selection of bifunctional RNA aptamers by chimeric SELEX, as described by Burke & Willis (1998), RNA, 4:1165-1175, selection using particle-bound ligands magnetic as described by Murphy et al. (2003) Nucleic Acids Res., 31:e110, an automatic SELEX technique described by Eulberg et al. (2005) Nucleic Acids Res., 33(4):e45, and a technique similar to SELEX 25 to obtain aptamers created against recombinant molecules expressed on cell surfaces, as described by Ohuchi et al. (2005) Nucleic Acid Symposium Series, 49:351 -352. Selection can begin with a random set of RNAs, from a partially structured set of RNAs (see, for example, Davis & Szostak (2002) Proc. Natl. Acad. Sci. USA, 99: 11616-11621), or from of a set of degenerate RNAs (see, for example, Geiger etal. (1996) Nucleic Acids Res., 24: 1029-1036). Models of secondary structure, folding, and hybridization behavior for a given RNA sequence can be predicted using algorithms, for example, as described by Zuker (2003) Nucleic Acids Res., 31: 3406-3415. In this way, aptamers for a given ligand can be designed de novo using appropriate selection. A non-limiting example of aptamer design and selection is described in detail in Weill et al. (2004) Nucleic 5 Acids Res., 32:5045-5058, which describes the isolation of several ATP-binding aptamers and secondary selection of aptamers that bind cordicepin (3' deoxyadenosine). Another non-limiting example of aptamer design is given in Huang & Szostak (2003) RNA, 9:1456-1463, which describe the in vitro evolution of new aptamers with new specificities and 10 new secondary structures from an initial aptamer. Ligands: ligands useful in the invention may include amino acids or their biosynthetic or catabolic intermediates, peptides, proteins, glycoproteins, lipoproteins, carbohydrates, fatty acids and other lipids, steroids, terpenoids, hormones, nucleic acids, aromatics, alkaloids, natural products or compounds synthetics (e.g., dyes, pharmaceuticals, antibiotics, herbicides), inorganic ions, and metals, into any short molecule (or part of a molecule) that can be recognized and be bound by a nucleic acid secondary structure by a mechanism not primarily based on Watson-Crick matching. Thus, 20 the recognition and binding of ligand and aptamer is analogous to that of antigen and antibody, or biological effector and receptor. Ligands may include single molecules (or part of a molecule), or a combination of two or more molecules (or parts of a molecule), and may include one or more macromolecular complexes (e.g., polymers, lipid bilayers, liposomes, cell membranes , or other cellular structures, or cellular surfaces). See, for example, Plummer et al. (2005) Nucleic Acids Res., 33:5602-5610, who describe selection of aptamers that bind to a small surface molecule-protein composite; Zhuang et al. (2002) J. Biol. Chem., 277:13863-13872, which describe the association of insect midgut receptor proteins with lipid rafts, which affects the binding of insecticidal endotoxins from Bacillus thuringiensis; and Homann & Goringer (1999) Nucleic Acids Res., 27:2006-2014, who describe aptamers that bind to live trypanosomes.

Non-limiting examples of specific ligands include vitamins such as coenzyme B12 and thiamine pyrophosphate, flavin mononucleotide, guanine, adenosine, S-adenosylmethionine, S-adenosylhomocysteine, coenzyme A, lysine, tyrosine, dopamine, glucosamine-6-phosphate, caffeine, theophylline, antibiotics such as chloramphenicol and neomycin, herbicides such as glyphosate and dicamba, proteins that include viral or phage coat proteins and epidermal or digestive tract surface proteins, and RNAs that include viral RNA, transfer RNAs (t- RNAs), ribosomal RNA (rRNA), and RNA polymerases such as RNA-dependent RNA polymerase (RdRP). Suitable ligands for use in this invention include insect midgut brush border receptor proteins that are recognized by Bacillus thuringiensis insecticidal endotoxins. See, for example, Knight et al. (1995) J. Biol. Chem., 270:17765-17770, and Gill et al. (1995) J. Biol. Chem., 270:27277-27282, which describe the isolation, identification, and cloning of examples of such receptor proteins; Gomez et al. (2001) J. Biol. Chem., 276:28906-28912, and Daniel et al. (2002) Appl. Env. Microbiol., 68:2106-2112, which describe techniques for identifying binding epitopes of such receptor proteins and for studying their binding affinities; Jurat-Fuentes & Adang (2001) Appl. Env. Microbiol., 67:323-329, and Jurat-Fuentes et al. (2001), Appl. Env. Microbiol., 67:872-879, which describe endotoxin receptor binding assays involving binding measured by both membrane blots and surface plasmon resonance of brush border membrane vesicles to endotoxin. Other examples of suitable ligands to which RNA aptamers of the invention bind include steroid receptors, such as estrogen receptors, androgen receptors, retinoid receptors, and ecdysone receptors (see, for example, Saez et al. (2000) Proc. Natl. Acad. Sci. USA, 97:1451214517. A class of RNA aptamers useful in the invention are ''. thermoswitches" that do not bind to a ligand but are thermally responsive, that is, the conformation of the aptamer is determined by temperature. See, for example, Table 3 in Mandai & Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451 -463. An aptamer can be described by its binding state, that is, whether the aptamer is bound (or unbound) to its respective ligand The binding site (or three-dimensional binding domain or domains. ) of an aptamer can be described as occupied or unoccupied by the ligand. Similarly, a population of a given aptamer can be described by the fraction of the population that is on or off the ligand. The affinity of an aptamer for its ligand can be described in terms of the rate of association (binding) of the aptamer with the ligand and the rate of dissociation of the ligand from the aptamer, for example, by the equilibrium association constant (K) or its reciprocal, the affinity constant (Ka) as is well known in the art. These rates can be determined by methods similar to those commonly used to determine the binding kinetics of ligands and receptors or antigens and antibodies, such as, but not limited to, equilibrium assays, competition assays, surface plasmon resonance, and models. predictive. The affinity of an aptamer for its ligand can be selected, for example, during in vitro evolution of the aptamer, or further modified by changes to the aptamer's primary sequence, where such changes can be guided by binding energy calculations or algorithms. for example, as described by Zuker (2003) Nucleic Acids Res., 31:3406-3415 or Bayer & Smolke (2005) Nature Biotechnol., 23:337-343. The binding state of an aptamer preferably at least partially determines the secondary structure (e.g., the formation of double-stranded or single-stranded regions) and the three-dimensional conformation of the aptamer. In embodiments where the DNA capable of being transcribed further includes DNA that transcribes regulatory RNA capable of regulating the expression of a target sequence, the binding state of the aptamer aiosterically affects the conformation of the regulatory RNA and thus the ability of the regulatory RNA to regulate expression of the target sequence.

In a preferred embodiment, the aptamer (transcribed RNA) is flanked by DNA that transcribes RNA capable of forming double-stranded RNA (dsRNA). In some of these embodiments, the dsRNA is processed by an RNAi mechanism (siRNA or miRNA), whereby the aptamer is cleaved from the rest of the transcript. In other particularly preferred embodiments, the two transcribed RNA regions flanking the aptamer at least partially form a double-stranded RNA stem between them, wherein the aptamer serves as a "spacer" or "handle" in a "stem" structure. stem-loop"; such an arrangement is expected to increase the stability or half-life of the transcript in a manner analogous to that observed for DNA (see, for example, Di Giusto & King (2004) J. Biol. Chem., 279:46483- 46489). Transgenic plants that have in their genome DNA that transcribes such aptamers that have increased stability are particularly desirable, for example, where the aptamer functions to inhibit or kill a pathogen or pest of the transgenic plant: in many. embodiments, the DNA capable of being transcribed further includes DNA that transcribes regulatory RNA capable of regulating the expression of a target sequence in which the regulation of the target sequence is dependent on the conformation of the regulatory RNA, and the conformation of the regulatory RNA is affected allosterically by the binding state of the RNA aptamer. Such combinations of an aptamer with a regulatory RNA domain are commonly known as "riboswitches". The regulatory RNA is typically downstream of the aptamer, but the two domains can overlap; see, for example, Najafi-Shoushtari & Famulok (2005) RNA, 11:15141520, which describes a hairpin ribozyme that includes an aptamer domain and is competitively regulated by flavin mononucleotide and an oligonucleotide complementary to the aptamer domain. In some 25 embodiments, the regulatory RNA is operably linked to the target sequence, and acts "in cis". In other embodiments, the regulatory RNA is not operatively linked to the target sequence and acts "in trans". Any target sequence may be chosen, including one or more target sequences selected from a gene native to the transgenic plant of the invention, a transgene in the transgenic plant, and a gene native to a pest or pathogen of the transgenic plant. The target sequence may include a sequence that expresses a gene of interest (e.g., an RNA that encodes a protein), or a sequence that suppresses a gene of interest (e.g., an RNA that is processed to an siRNA or miRNA that by in turn suppresses the gene of interest). The target sequence may be a sequence capable of being translated (coding), or it may be a non-coding sequence (such as a non-coding regulatory sequence), or both. The target sequence may include at least one eukaryotic target sequence, at least one non-eukaryotic target sequence, or both. A target sequence may include any sequence from any species (including, but not limited to, non-eukaryotes, such as bacteria and viruses; fungi, plants; including monocots and dicots, such as crop plants, ornamental plants, and non- domesticated or wild; invertebrates such as arthropods, annelids, nematodes, and mollusks; and vertebrates such as amphibians, fish, birds, domestic or wild mammals, and even humans. Target Genes.”

In riboswitch embodiments that include an aptamer and a regulatory RNA, the riboswitch regulates expression of the target sequence by any suitable mechanism. A non-limiting mechanism is transcriptional regulation by ligand-dependent formation of an intrinsic terminator stem (an extended "stem-loop" structure typically followed by a series of 6 or more U residues) that causes RNA polymerase to abort transcription , for example, before the complete mRNA is formed. In "off" riboswitches, in the absence of sufficient ligand, the unbound aptamer domain allows the formation of an "antiterminator stem", which prevents the formation of the intrinsic terminator stem and thus allows transcription to occur; thus, the default state of the riboswitch is "on" (i.e., transcription occurs normally) and ligand must be added to turn the riboswitch off. In "tethered" riboswitches that use this mechanism, the aptamer domain must be in the bound conformation (ligand occupied) to allow the formation of the "antiterminator stem" and allow transcription. Another mechanism is translational regulation, where ligand binding causes structural changes in full-length mRNAs and thus allows (or prevents) ribosomes from binding to the ribosome binding site (RBS); the formation of an "anti-anti-RBS" trunk and an "anti-RBS" trunk is also mutually exclusive. In "on" riboswitches that use this mechanism, the absence of the ligand allows the formation of an anti-anti-RBS, and thus a structurally unencumbered RBS to which the ribosome can bind. A combination of transcriptional and translational regulation is also possible. For a detailed discussion of regulatory mechanisms, see Mandai & Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463.

In some embodiments, the regulatory RNA includes a ribozyme, for example, a self-cleaving ribozyme, a hammerhead ribozyme, or a hairpin ribozyme. Certain embodiments of regulatory RNA include RNA sequences that are complementary or substantially complementary to the target sequence. A non-limiting example is where the regulatory RNA includes an antisense segment that is complete. mentary or substantially complementary to the target sequence. See, for example, Bayer & Smolke (2005) Nature Biotechnol., 23:337-343, where the regulatory RNA 15 includes both an antisense segment complementary to the target sequence, and a sense segment complementary to the antisense segment, in which The antisense segment and the sense segment are capable of hybridizing to each other to form an intramolecular double-stranded RNA.

In some embodiments, regulation of a target sequence involves Watson-Crick base pairing of the regulatory RNA to the target sequence (e.g., in transacting embodiments see, e.g., Bayer & Smolke (2005) Nature Biotechnol., 23:337 -343). Particularly in the case of such transacting embodiments, suitable target sequences include the target genes described under the heading "Target Genes." A target sequence of interest can be achieved more specifically by designing the regulatory RNA to include regions substantially non-identical to a sequence not Target sequences can include any gene for which expression is preferably not modified, both in a plant that transcribes the recombinant DNA construct and in organisms that can contact RNA transcribed from the DNA construct. Recombinant A non-target sequence may include any sequence from any species (including, but not limited to, non-eukaryotes such as bacteria, and viruses; fungi, plants; including monocots and dicots, such as crop plants, plants. ornamentals, and non-domesticated or wild plants; invertebrates such as arthropods, annelids, nematodes, and molluscs; and vertebrates such as amphibians, fish, birds, domestic or wild mammals, or even humans).

One embodiment provides a transgenic plant that has in its genome a recombinant DNA construct that includes DNA capable of being transcribed that includes DNA that transcribes RNA capable of binding a ligand, wherein the construct confers male sterility or inducible fertility or chemically suppressible for hybridization. Preferred examples use a riboswitch that contains an aptamer that binds to a ligand that is an already registered substance, for example, an approved herbicide. In a non-limiting example, a transgenic plant that carries a male sterility gene under the control of a male-specific promoter and a glyphosate "off" riboswitch is male sterile unless glyphosate is applied. In contrast, a transgenic plant that carries a male sterility gene under the control of a male-specific promoter and a glyphosate "turned on" riboswitch is male sterile only when glyphosate is applied.

One application of the invention is to provide a ligand-activated, herbicide-resistant system for preserving gene identity ("gene blocking") as well as maintaining voluntary, herbicide-resistant control. In one embodiment, the DNA sequence encoding a "turned on" riboswitch is inserted into an expression cassette that contains as the target sequence "CP4", a selectable marker that confers resistance to glyphosate, epsps~cp4 (5-enolpyruvylshikimate-3 -phosphate synthase from Agrobacterium tumefaciens strain CP4), to conditionally express CP4 in transgenic plants. Transgenic plants carrying the riboswitch-controlled CP4 expression cassette express CP4 only in the presence of the ligand, which is applied (e.g., by a foliar spray) to the plant via a specific glyphosate formulation containing the ligand. When applied, the formulated glyphosate herbicide activates the transcription/translation of CP4 and makes the transgenic plant resistant to glyphosate. Transgenic plants are susceptible to generic glyphosate formulations that do not contain the ligand. Likewise, this approach can be applied to any other herbicide/herbicide resistance gene combinations, for example, dicamba/dicamba degrading oxygenase combinations, or antibiotic/antibiotic resistance gene.

In another embodiment, the target sequence is a gene endogenous to a given species, such as a given plant (such as, but not limited to, agriculturally or commercially important plants, including monocots and dicots) and the non-target sequence may be, for example, a gene from a non-target species, such as another plant species or a gene from a virus, fungus, bacteria, invertebrate, or vertebrate or even a human being. A non-limiting example is where it is desirable to design the aptamer, or the regulatory RNA, or both, in order to modify the expression of a target sequence that is endogenous to the gene for a single species (e.g., corn rootworm). Western, Diabrotica virgifera virgifera Le- 15 Conte) but does not modify the expression of a non-target sequence such as genes from related, even closely related, species (e.g., Northern rootworm, Diabrotica barberi Smith & Lawrence, or Southern corn rootworm, Diabrotica undecimpunctata).

In other embodiments (e.g., where it is desirable to modify the expression of a target sequence among multiple species), it may be desirable to design the aptamer, or the regulatory RNA, or both, to modify the expression of a target sequence common to multiple species. species in which expression of the target sequence must be modified. In this way, the aptamer, or the regulatory RNA, or both, can be selected to be specific to a taxon (e.g., specific to a genus, family, or even a larger taxon such as a phylum, e.g., arthropods). ), but not for other taxa (e.g. plants or vertebrates or mammals). In a non-limiting example of this embodiment, a regulatory RNA may be selected to target pathogenic fungi (e.g., a Fusarium spp.), but not target any gene sequences of beneficial fungi (e.g., beneficial mycorrhizal fungi). from soil).

In another non-limiting example of this embodiment, the aptamer, or the regulatory RNA, or both, for regulating expression in corn rootworm can be selected to be specific to all members of the genus Diabrotica. For example, a regulatory RNA that includes a gene suppression element targeting Diabrotica (e.g., antisense RNA, double-stranded RNA, microRNA, or tandem RNA repeats) can be selected to target any Diabrotica gene sequence. beneficial coleoptera (e.g., beneficial coccinellid beetles, commonly known as ladybugs) or other beneficial insect species. The necessary degree of specificity of a regulatory RNA that includes a gene suppression element (e.g., antisense RNA, double-stranded RNA, microRNA, or tandem RNA repeats) for suppression of a target sequence depends on several factors. For example, where the gene suppression element includes double-stranded RNA (dsRNA), factors may include the size of the smallest dsRNA fragments expected to be produced by the action of Dicer, and the relative importance of decreasing the potential of dsRNA's to suppress non-target sequences. For example, where dsRNA fragments are expected to be 21 base pairs in size, a particularly preferred embodiment may be to include in the regulatory RNA, a sequence capable of forming dsRNA and encoding regions substantially non-identical to a non-target sequence, such as as regions within which each contiguous fragment that includes at least 21 nucleotide pairings of less than 21 (e.g., of less than 21, or of less than 20, or of less than 19, or of less than 17) of the 21 contiguous nucleotides of a non-target sequence. In another embodiment, regions substantially non-identical to a non-target sequence include regions within which each contiguous fragment that includes at least 19 nucleotide pairings of less than 19 (e.g., less than 19, or less than 18, or less than 17, or less than 16) of the 19 contiguous nucleotides of a non-target sequence.

In some embodiments, it may be desirable to design the aptamer, the regulatory RNA, or both, to include regions predicted not to generate undesirable polypeptides, e.g., by screening the aptamer, the regulatory RNA, or both, for sequences that may encode polypeptides. - known undesirable peptides or close homologues thereof. Undesirable polypeptides include, but are not limited to, polypeptides homologous to known allergenic polypeptides and polypeptides homologous to known polypeptide toxins. Publicly available sequences encoding such potentially allergenic peptides are available, for example, the Food Allergy Research and Re-source Program (FARRP) allergen database (available at allergenonline.com) or the Biotechnology Information for Food Safety databases Databases (available at www.iit.edu/~sgendel/fa.htm) (see also, for example, Gendel (1998) Adv. Food Nutr. Res.f 42:63-92). Undesirable sequences may also include, for example, those polypeptide sequences identified as known toxins or as potential or known allergens and contained in publicly available databases such as GenBank, EMBL, SwissProt, and others, which are searchable by the Entrez system ( www.ncbi.nih.gov/Entrez). Non-limiting examples of potentially allergenic peptide sequences include soy glycinin, peanut oleosin and agglutinin, wheat glutenins, casein, lactalbumin, and milk lactoglobulin. bovine and tropomyosin from various molluscs (allergenonline.com). 20 Non-limiting examples of undesirable potentially toxic peptides include tetanus toxin tetA from Clostridium tetani, diarrheal toxins from Staphylococcus aureus, and venoms such as conotoxins from Conus spp. and neurotoxins from arthropods and reptiles (www.ncbi.nih.gov/Entrez).

In a non-limiting example, a proposed aptamer, regulatory RNA, or both, can be screened to eliminate those sequences capable of being transcribed that encode polypeptides with perfect homology to a known allergen or toxin for 8 contiguous amino acids, or with at least at least 35% identity for at least 80 amino acids; Such scans can be performed on any and all possible open reading phases in both directions, on potential open reading phases that begin with ATG, or on all possible open reading phases, regardless of whether they begin with an ATG or no. When a 'hit' or pairing is made, that is, when a sequence encoding a potential polypeptide with perfect homology to a known allergen or toxin for 8 consecutive amino acids (or at least about 35% identity for at least about 80 amino acids), is identified, the nucleic acid sequences that correspond to the hit can be avoided, eliminated, or modified when selecting sequences to be used in an RNA to silence a target gene.

Avoiding, eliminating, or modifying an unwanted sequence can be accomplished by any of a number of methods known to those skilled in the art. In some cases, the result may be new sequences that are believed not to exist naturally. For example, avoiding certain sequences can be achieved by splicing "clean" sequences into new chimeric sequences to be used in a gene suppression element. Where regulatory RNA includes double-stranded RNA (dsRNA) to silence a target gene, applicants recognize that in some dsRNA-mediated silencing, it is possible that dsRNA sequences that do not pair perfectly are effective in gene silencing. For example, it has been shown that mismatches near the center of a miRNA complementary site have stronger effects on miRNA gene silencing than pairings located more distally. See, for example, Figure 4 in Mallory et al. (2004) EMBO J., 23:3356-3364. In another example, it was reported that both the position of an unpaired base pair and the identity of the nucleotides forming the unpaired influence the ability of a given siRNA to silence a target sequence, and that non-adenine-cytosine pairings, in addition to the G:U wobble base pair, they were well tolerated (see Du et al. (2005) Nucleic Acids Fies., 33:16711677). Thus, a regulatory dsRNA that includes double-stranded RNA need not always have 100% sequence identity with the intended target sequence, but generally would preferably have substantial sequence identity with the intended target sequence, such as about 95%, about 90%, about 85%, or about 80% sequence identity with the intended target sequence. One skilled in the art would be able to judge the importance given to screening for regions predicted to be more highly specific to the first target sequence or predicted not to generate undesirable polypeptides, relative to the importance given to other criteria, such as, but not limited to, percentage of sequence identity with the first intended target sequence or the predicted gene silencing efficiency of a given sequence. For example, it may be desirable for a given regulatory RNA that includes double-stranded RNA for gene silencing to be active across multiple species, and therefore one of skill in the art may determine that it is more important to include in the regulatory RNA regions specific to the various species of interest. but less important to screen for regions predicted to have greater gene silencing efficiency or for regions predicted to generate undesirable polypeptides.

In many embodiments, the transgenic plant cell has in its genome recombinant DNA that includes DNA capable of being transcribed that includes: (a) DNA that transcribes an RNA aptamer capable of binding a ligand, and (b) DNA that transcribes Regulatory RNA capable of regulating the expression of a target sequence, in which regulation is dependent on the formation of regulatory RNA, and the conformation of said regulatory RNA is affected allosterically by the binding state of said aptamer. RNA. In these embodiments, binding of the aptamer to its ligand results in a specific change in the expression of the target sequence, which may be an increase or decrease in expression, depending on the design of the recombinant DNA.

In one embodiment, binding of the ligand to the RNA aptamer results in an increase in expression of the target sequence relative to expression in the absence of binding. In another embodiment, binding of the ligand to the RNA aptamer results in a decrease in expression of the target sequence relative to expression in the absence of binding.

Some modalities are characterized by "self-inducibility". In such an embodiment, ligand binding to the RNA aptamer results in an increase in expression of the target sequence relative to expression in the absence of binding, wherein the increase in expression results in a level of ligand sufficient to maintain the increase. of expression. In another embodiment, binding of the ligand to the RNA aptamer results in a decrease in expression of the target sequence relative to expression in the absence of binding, the decrease in expression resulting in a level of ligand sufficient to maintain the increase in expression.

Accordingly, another aspect of the invention is a method of modifying the expression of a gene of interest in a plant cell, which includes transcribing into a transgenic plant cell of the invention, or a plant, progeny plant, or seed or other tissue. derived from such a transgenic plant cell, recombinant or heterologous DNA capable of regulating the expression of a target sequence, wherein the regulation is dependent on the conformation of the regulatory RNA, and wherein the conformation of the regulatory RNA is affected allosterically by the state of binding of the RNA aptamer, whereby the expression of the gene of interest is modified relative to its expression in the absence of transcription of the recombinant DNA construct. Introns: As used herein, "intron" or "intron sequence" generally means non-coding DNA sequence of a natural gene, which retains in the recombinant DNA constructs of this invention, its native ability to be excised from pre-mRNA transcripts , for example, native intron sequences found associated with protein-coding regions of RNA, in which native introns undergo splicing, allowing exons to be assembled into mature mRNAs before the RNA leaves the nucleus. Such a removable intron has a 5' splice site and a 3' splice site. Introns can undergo "self-braiding" or not undergo "self-braiding" (i.e., requiring enzymes or a spliceosome for splicing to occur) and can be selected for different splicing efficiency. Suitable introns for use in constructs of the invention may be viral introns (e.g., Yamada et al. (1994) Nucleic Acids Res., 22:2532-2537), eukaryotic introns (including animal, fungal and plant introns), archaeal or bacterial introns (e.g., Belfort et al. (1995) J. Bacteríol., 177:3897-3903), or any naturally occurring or artificial DNA sequence (e.g., Yoshimatsu & Nagawa (1989) Science, 244 :1346-1348) with intron-like functionality in the plant in which the recombinant DNA construct of the invention is to be transcribed. Although essentially any intron can be used in the practice of this invention as a host for inserted DNA, particularly preferred are introns that increase expression in a plant or introns that are derived from a 5' untranslated leader sequence. When a recombinant DNA construct of the invention is used to transform a plant, introns originating in the plant may be especially preferred. Examples of especially preferred plant introns include a rice actin 1 intron (I-Os-Act1) (Wang et al. (1992) Mol. Cell Biol., 12:3399-3406; McEI-roy et al. ( 1990) Plant Cell, 2:163-171), an intron of corn heat shock protein (l-Zm-hsp70) (U.S. Patents 5,593,874 and 5,859,347), and an intron of corn alcohol dehydrogenase (l-Zm -adh1) (Callis etal. (1987) Genes Dev., 1:1183-1200). Other examples of introns suitable for use in the invention include the tobacco mosaic virus 5' leader sequence or "omega" leader 15 (Gallie & Walbot (1992) Nucleic Acids Res., 20:4631-4638), the Shrunken- 1 (Sh-1) (Vasil et al. (1989) Plant Physiol., 91:1575-1579), the maize sucrose synthase intron (Clancy & Hannah (2002) Plant Physiol., 130:918-929), the heat shock protein intron 18 (hsp18) (Silva et al. (1987) J. Cell Biol., 105:245), and the heat shock protein 82 kilodalton intron (hsp82) (Semrau et al. ( 1989) J. Cell Biol., 109, p. 39A, and Mettler et al. (May 1990) N.A.T.O. Institute on Molecular Biology, Elmer, Bavaria).

Gene expression elements: Recombinant DNA constructs may further include a gene expression element. Any gene or genes of interest may be expressed by the gene expression element, including coding or non-coding sequences, or both, and may include naturally occurring sequences or artificial or chimeric sequences or both. When the gene expression element encodes a protein, such constructs preferably include a functional terminator element to allow transcription and translation of the gene expression element. In embodiments that include inductively sterile transgenic plants, the gene expression element may include, or may be co-located with, messenger RNA encoding a herbicide tolerance protein.

In embodiments in which the recombinant DNA construct contains an intron, the construct may further include an element. gene expression to express at least one gene of interest, wherein the gene expression element is located adjacent to the intron. In other embodiments, the recombinant DNA construct includes an intron into which a gene of interest is inserted, wherein the gene expression element is also located within the intron; in such cases, the gene expression element can be operatively linked to a functional terminator element that is itself also within the intron. The gene of interest 10 to be expressed by the gene expression element may include at least one gene selected from the group consisting of a eukaryotic target gene, a non-eukaryotic target gene, a microRNA precursor DNA sequence. The gene of interest may include a single gene or multiple genes (such as multiple copies of a single gene, multiple alleles of a single gene, or multiple genes that include genes from multiple species).

In one embodiment, the gene expression element may include self-hydrolyzing peptide sequences, for example, located between multiple coding sequences for one or more polypeptides (see, for example, the 2A and "2A-like" self-cleavage sequences of various species, 20 including viruses, trypanosomes, and bacteria, described by Donnelly et al (2001), J. Gen. Viro!., 82:1027-1041). In another embodiment, the gene expression element may include ribosomal "jumping" sequences, for example, located between multiple sequences for one or more polypeptides (see, for example, the 2A ribosomal "jumping" sequences of the foot-and-mouth disease aphthovirus (FMDV) described by Donnelly et al. (2001), J. Gen. Virol., 82:1013-1025). A gene of interest may include any coding or non-coding sequence from any species (including, but not limited to, non-eukaryotes such as bacteria and viruses; fungi; plants, including monocotyledons and dicots, such as crop plants, ornamental plants, and non-domesticated or wild plants; invertebrates such as arthropods, annelids, nematodes, and molluscs; and vertebrates such as amphibians, fish, birds, and mammals. of gene expression include, but not limited to, 5' untranslated regions, promoters, enhancers, or other non-coding transcriptional regions, 3' untranslated regions, terminators, introns, microRNAs, microRNA precursor DNA sequences, small interfering RNAs, RNA components of ribosomes or ribozymes, small nucleolar RNAs, and other non-coding RNAs. Non-limiting examples of a gene of interest further include, but are not limited to, sequence capable of being translated ( coding), such as genes encoding transcription factors and genes encoding enzymes involved in the biosynthesis or catabolism of molecules of interest (such as amino acids, fatty acids and other lipids, sugars and other carbohydrates, biological polymers, and metabolites of mixed biosynthetic origin). A gene of interest may be a gene native to the plant in which the recombinant DNA construct of the invention is to be transcribed, or it may be a non-native gene. A gene of interest may be a marker gene, for example, a selectable marker gene encoding a gene.de. antibiotic, antifungal, or herbicide resistance or a marker gene that encodes an easily detectable characteristic (e.g., phytoene synthase or other genes that confer a particular pigment to the plant), or a gene that encodes a detectable molecule, such as a fluorescent protein, luciferase, or a single polypeptide or nucleic acid "flag" detectable by protein or nucleic acid detection methods, respectively). Selectable markers are genes of interest of particular utility in identifying successful processing of constructs of the invention.

In some embodiments of the invention, recombinant DNA constructs are designed to suppress at least one endogenous or exogenous gene and to simultaneously express at least one exogenous gene. In a non-limiting example, the recombinant DNA construct includes a gene suppression element for suppressing an endogenous lysine ketoglutarate reductase/saccharopine dehydrogenase (LKfi/SDH) gene (from maize), a gene expression element for expressing a exogenous (bacterial) dihydrodipicolinic acid synthase protein, and a DNA that transcribes RNA that includes: (a) at least one exogenous miRNA recognition site recognizable by a mature miRNA that is specifically expressed in plant reproductive tissue; and (b) messenger RNA encoding epsps-CP4, in which the mature miRNA specifically suppresses the expression of epsps-CP4 in reproductive tissue, and in which sterility of the transgenic plant is inducible by application of glyphosate to the plant. Such a construct would be especially useful for providing corn with increased lysine levels and general glyphosate tolerance, where corn sterility is inducible by application of glyphosate at the appropriate developmental time(s). In another non-limiting example, the recombinant DNA construct includes a gene suppression element for suppressing a corn rootworm gene, and DNA that transcribes RNA that includes: (a) at least one exogenous miRNA recognition site recognizable by a mature miRNA that is specifically expressed in plant reproductive tissue; and (b) messenger RNA encoding epsps-CP4, in which the mature miRNA specifically suppresses the expression of epsps-CP4 in reproductive tissue, and in which the sterility of the transgenic plant is inducible by the application of glyphosate to the plant. Such a construct would be especially useful for providing corn with corn rootworm resistance and general glyphosate tolerance in which corn sterility is inducible by application of glyphosate at the appropriate developmental time(s).

T-DNA Edges: T-DNA edges refer to the DNA sequences or DNA regions that define the beginning and end of an Agrobacterium T-DNA (tumor DNA) and function in cis to transfer of T-DNA into a plant genome by Agrobacterium-mediated transformation (see, for example, Hooykaas & Schilperoort (1992) Plant Mol. Biol., 19:15-38). Various embodiments include recombinant DNA constructs without T-DNA borders, a single T-DNA border, or more than one T-DNA border. For example, in embodiments where the recombinant DNA construct includes an intron into which a gene suppression element is inserted, the intron is preferably located between a pair of T-DNA edges, which may be a group of T-DNA edges. left and right, a group of two left T-DNA borders, or a group of two right T-DNA borders. In another embodiment, the recombinant DNA construct includes a single T-DNA border, an intron-inserted gene suppression element, and at least one gene expression element. Terminators: Many embodiments, particularly where the recombinant DNA construct is designed to be transcribed into messenger RNA, include both a promoter element and a functional terminator element that includes a functional polyadenylation signal and a polyadenylation site, allowing the transcribed RNA from the recombinant DNA construct is polyadenylated and processed for transport into the cytoplasm.

In general embodiments as described under the heading "Constructs (i.e. of Recombinant DNA that Include MicroRNA Recognition Sites), a functional terminator element may be present or absent. In embodiments where a functional terminator element is absent, at least one of a functional polyadenylation signal and a functional polyadenylation site is absent. In other embodiments, a 3' untranslated region is absent. In these cases, the recombinant DNA construct is transcribed as non-polyadenylated RNA and is preferably not transported into. of the cytoplasm.

Spacer DNA: Spacer DNA segments may include virtually any DNA (such as, but not limited to, transcribeable DNA sequence encoding a gene of interest, transcribeable DNA sequence encoding a marker or reporter gene; Transcribeable DNA derived from an intron, which on transcription can be excised from the resulting transcribed RNA; transcribed DNA that encodes RNA that forms a structure such as a loop or "stem" or an aptamer capable of binding to an intron. specific ligand; DNA that can undergo splicing such as introns and ribozymes that undergo "self-twisting"; DNA capable of being transcribed that encodes a sequence for detection by nucleic acid hybridization, amplification, or sequencing; ). Spacer DNA can be found, for example, adjacent to a gene expression element. In some embodiments, the spacer DNA is itself the sense or antisense sequence of the target gene. In some preferred embodiments, the RNA transcribed from spacer DNA (e.g., a large loop of antisense sequence from the target gene or an aptamer) assumes a secondary structure or three-dimensional configuration that confers on the transcript a desired characteristic, such as stability. increased, increased in vivo half-life, or cell or tissue specificity.

Making and Using Recombinant DNA Constructs

The recombinant DNA constructs of the present invention can be made by any method suitable for the intended application, taking into account, for example, the type of expression desired and the convenience of use in the plant in which the construct is to be transcribed. Common methods for making and using DNA constructs and vectors are well known in the art and described in detail, for example, in textbooks and laboratory manuals including Sambrook & Russell, "Molecular Cloning: A Laboratory Manual" (third edition), Cold Spring Harbor Laboratory Press, NY, 2001. An example of useful technology for constructing DNA constructs and vectors for transformation is described in US Patent Application Publication 2004/0115642 A1. DNA constructs can also be constructed using GATEWAY™ cloning technology (available from Invitrogen Life Technologies, Carlsbad, CA), which uses the site-specific LR recombinase cloning reaction of the Integrase/aft system for lambda bacteriophage vector construction, rather than restriction endonucleases and ligases. The LR cloning reaction is described in U.S. Patents 5,888,732 and 6,277,608 and in U.S. Patent Application Publications 2001/283529, 2001/282319 and 2002/0007051. The GATEWAY™ Cloning Technology Instruction Manual, which is also provided by Invitrogen, provides summary instructions for routine cloning of any desired DNA into a vector comprising operable plant expression elements. Another alternative vector manufacturing method employs ligation-independent cloning as described by Aslandis et al. (1990) Nucleic Acids Res., 18:6069-6074 and Rashtchian et al. (1992) Biochem., 206:91-97, where a DNA fragment with single-stranded 5' and 3' ends is ligated to a desired vector that can then be amplified in vivo.

In certain embodiments, the DNA sequence of the recombinant DNA construct includes a sequence that has been codon optimized for the plant in which the recombinant DNA construct is to be expressed. For example, a recombinant DNA construct to be expressed in a plant may have all or part of its sequence (e.g., the first gene suppression element or the gene expression element) codon optimized for expression in a plant by methods known in the art. See, for example, U.S. Patent 5,500,365 for a description of codon optimization for plants; see also De Amicis & Marchetti (2000) Nucleic Acid Res., 28:3339-3346.

TRANSGENIC PLANT CELLS AND TRANSGENIC PLANTS

A further aspect of this invention provides a transgenic plant cell that has in its genome any of the recombinant DNA constructs provided by this invention. A transgenic plant containing transgenic plant cells of this invention is further provided. The transgenic plant of the invention includes plants at any stage of development and includes a regenerated plant prepared from the transgenic plant cells claimed herein or a progeny plant (which may be a natural or hybrid progeny plant) of the regenerated plant or seed of such a transgenic plant. Also provided and claimed is a transgenic seed that has in its genome any of the recombinant DNA constructs provided by this invention.

One embodiment includes a transgenic plant that has in its genome a recombinant DNA construct that transcribes RNA that includes: (a) at least one exogenous miRNA recognition site recognizable by a mature miRNA that is specifically expressed in reproductive tissue of the plant (or that is, a plant prepared from a transgenic plant cell); and (b) messenger RNA encoding a protein that confers tolerance to a herbicide; wherein the mature miRNA specifically suppresses expression of the protein in reproductive tissue, and wherein the sterility of a transgenic plant prepared from the transgenic plant cell is inducible by application of the herbicide to the plant.

The transgenic plant cell may be an isolated plant cell (e.g., individual plant cells or cells grown in or on an artificial culture medium) or may be a plant cell in undifferentiated tissue (e.g., callus or any plant cell aggregate). The transgenic plant cell may be a plant cell in at least one differentiated tissue selected from the group consisting of leaf (e.g., petiole and blade), root, stem (e.g., tuber, rhizome, stolon, bulb and corm), stem (e.g. xylem and phloem), wood, seed, fruit (e.g. nut, grain, fresh fruit) and flower (e.g. stamen, filament, anther, pollen, carpel, pistil, ovary, ovules ).

The transgenic plant cell or transgenic plant of the invention can be any plant cell or plant of interest, both transiently transformed and stably transformed plant cells are encompassed by this invention. Stably transformed transgenic plants are particularly preferred. In many preferred embodiments, the transgenic plant is a fertile transgenic plant from which the seed can be harvested and the invention further claims transgenic seed from such transgenic plants, wherein the seed preferably also contains the recombinant construct of this invention.

Making and Using Transgenic Plant Cells and Transgenic Plants

When a recombinant DNA construct is used to produce a transgenic plant cell, transgenic plant or transgenic seed of this invention, the transformation can include any of the well-known and demonstrated methods and compositions. Suitable methods for plant transformation include virtually any method by which DNA can be introduced into a cell, such as by direct release of DNA (e.g., PEG-mediated protoplast transformation, by electroporation, by agitation with silicon carbide fibers). , and by acceleration of particles coated with DNA), by transformation mediated by Agrobacterium, by viral vectors and others, etc. A preferred method of plant transformation is bombardment with micro-projectiles, for example, as illustrated in U.S. Patents 5,015,580 (soybeans), 5,550,318 (corn), 5,538,880 (corn), 6,153,812 (wheat). , 6,160,208 (corn), 6,288,312 (rice) and 6,399,861 (corn) and 6,403,865 (corn). Another preferred method of plant transformation is Agrobacterium-mediated transformation. In a preferred embodiment of the invention, the transgenic plant cell of the invention is obtained by transformation using Agrobacterium that contains a binary Ti 5 plasmid system, wherein the Agrobacterium carries a first Ti plasmid and a second, chimeric plasmid that contains at least one T-DNA border of a wild-type Ti plasmid, a promoter functional in the transformed plant cell and operably linked to a gene suppression construct of the invention. See, for example, the binary system described in U.S. Patent 5,159,135. See also De Framond (1983) Biotechnology, 1:262-269; and Hoekema et al., (1983) Nature, 303:179. In such a binary system, the smallest plasmid, containing the T-DNA border or borders, can be conveniently constructed and manipulated in a suitable alternative host, such as E coli, and then transferred to Agrobacterium. Detailed procedures for Agrobacterium-mediated transformation of plants, especially crop plants, include, for example, the procedures described in U.S. Patents 5,004,863, 5,159,135, and 5,518,908 (cotton); 5,416,011, 5,569,834, 5,824,877 and 6,384,301 (soy); 5,591,616 (corn); 5,981,840 (corn); 5,463,174 (brassicas). Similar methods have been reported for many plant species, both dicotyledonous and monocotyledonous, including, among others, peanut (Cheng et al. (1996) Plant Cell Rep., 15: 653); asparagus (Bytebier et al. (1987) Proc. Natl. Acad. Sci. U.S.A., 84:5345); barley (Wan & Lemaux (1994) Plant Physiol., 104:37); rice (Toriyama et al. (1988) Bio/Technology, 6:10; Zhang et al. (1988) Plant Cell Rep., 7:379; wheat (Vasil et al. (1992) Bio/Technology, 10:667; Becker etal. (1994) Plant J., 5:299), alfalfa (Masoud et al. (1996) Transgen. Res., 5:313); :426-431). CaMV35S. Transgenic plant cells and transgenic plants can also be obtained by transformation with other vectors, such as, but not limited to, viral vectors (e.g., tobacco etch potyvirus (TEV), barley striatum mosaic virus (BSMV). ) and viruses referenced in Edwardson & Christie, "The Potyvirus Group: Monograph No. 16, 1991, Agric. Exp. Station, Univ of Florida), plasmids, cosmids, YACs (yeast artificial chromosomes), BACs ( bacterial artificial chromosomes) or any other suitable cloning vector, when used with an appropriate transformation protocol, e.g., bacterial infection (e.g. with Agrobacterium as described above), binary bacterial artificial chromosome constructs, direct release of DNA (e.g. example, through PEG-mediated transformation, desiccation/inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers, and microprojectile bombardment). It will be clear to one skilled in the art that various transformation methodologies can be used and modified to produce stable transgenic plants from any number of plant species of interest. Transformation methods for providing transgenic plant cells and transgenic plants containing stably integrated recombinant DNA are preferably practiced in tissue culture medium and in a controlled environment. "Medium" refers to the various nutrient mixtures that are used to grow cells in vitro, that is, outside the intact living organism. Recipient target cells include, but are not limited to, meristem cells, callus, immature embryos or parts of embryos, gamete cells such as microspores, pollen, sperm and egg cells. Any cell from which a fertile plant can be regenerated is contemplated as a useful recipient cell for practicing the invention. Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, and the like. Those cells that are capable of proliferating as callus can serve as receptor cells for genetic transformation. Practical methods and transformation materials for producing the transgenic plants of this invention (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are described, for example, in U.S. Patents 6,194,636 and 6,232 526 and in U.S. Patent Application Publication 2004/0216189.

In common transformation practice, DNA is introduced into only a small percentage of target cells in any transformation experiment. Marker genes are generally used to provide an effective system for identifying those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers that confer resistance to a selective agent, such as an antibiotic or herbicide. Any of the antibiotics or herbicides to which a plant cell may be resistant can be a useful agent for selection. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the gene that confers resistance is integrated and expressed at levels sufficient to allow the cell to survive.

Cells can be further tested to confirm stable integration of the recombinant DNA. Commonly used selective marker genes include those that confer resistance to antibiotics such as kanamycin or paromomycin (nptll), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glyphosinate (bar or pat ) and glyphosate (EPSPS). Examples of useful selective marker genes and selection agents are illustrated in U.S. Patents 5,550,318, 5,633,435, 5,780,708 and 6,118,047. Traceable markers or reporters, such as markers that provide an ability to visually identify transformants, may also be employed. Non-limiting examples of useful traceable markers include, for example, a gene that expresses a protein that produces a detectable color by action on a chromogenic substrate (e.g., beta-glucuronidase (GUS) (uidk) or luciferase (luc) or that is detectable by itself, such as green fluorescent protein (GFP) (gfp) or an immunogenic molecule. Those skilled in the art will recognize that many other useful markers or reporters are available for use.

Detection or measurement of the resulting change in expression of a target gene (or concomitant expression of a gene of interest) obtained by transcription of the recombinant construct in the transgenic plant of the invention can be achieved by any suitable means, including protein detection methods (e.g. example, western blots, ELISAs and other immunochemical methods), enzymatic activity measurements or nucleic acid detection methods (e.g. Southern blots, northern blots, PCR, RT-PCR, fluorescent in situ hybridization). Such methods are well known to those skilled in the art as evidenced by the various manuals available; see, for example, Joseph Sambrook & David W. Russell, "Molecular Cloning: A Laboratory Manual" (third edition), Cold Spring Harbor Laboratory Press, NY, 2001; Frederick M. Ausubel et al. (editors) "Short Protocols in Molecular Biology" (fifth edition), John Wiley and Sons, 2002; John M. Walker (editor) “Protein Protocols Handbook” (second edition), Humana Press, 2002; and Leandro Pena (editor) “Transgenic Plants: Methods and Protocols”, Humana Press, 2004. Other methods suitable for detecting or measuring protein resulting change in expression of a target gene (or concomitant expression of a gene of interest) obtained by transcription of the recombinant DNA in the transgenic plant of the invention includes the measurement of any other characteristic that is a direct or representative indication of expression of the target gene ( or concomitant expression of a gene of interest) in the transgenic plant in which the recombinant DNA is transcribed, relative to one in which the recombinant DNA is not transcribed, e.g., gross or microscopic morphological characteristics, growth rates, yield, rates of reproduction or recruitment, resistance to pests or pathogens, or resistance to biotic or abiotic stress (e.g., water stress, salt stress, nutrient stress, heat or cold stress). Such methods may use direct measurements of a phenotypic trait or surrogate assays (for example, in plants, these assays include plant part assays such as leaf or root assays to determine tolerance to abiotic stress). The recombinant DNA constructs of the invention can be added with other recombinant DNA to confer additional characteristics (e.g., in the case of transformed plants, characteristics include herbicide resistance, pest resistance, tolerance to cold germination, tolerance to lack of water and the like), for example, by the expression or suppression of other genes. Constructs for the coordinated decrease or increase of gene expression are described in U.S. Patent Application Publication 2004/0126845 A1. Fertile, transgenic plant seeds can be collected and used to cultivate progeny generations, including hybrid generations, of transgenic plants of this invention that include the recombinant DNA construct in their genomes. Thus, in addition to directly transforming a plant with a recombinant DNA construct, transgenic plants of the invention can be prepared by crossing a first plant that has the recombinant DNA with a second plant that does not have the construct. For example, recombinant DNA can be introduced into a plant line that is amenable to transformation to produce a transgenic plant, which can be crossed with a second plant line to introduce the recombinant DNA into the resulting progeny. A transgenic plant of the invention with a recombinant DNA (which effects alteration of a target gene) can be crossed with a plant line that has another recombinant DNA that confers one or more additional characteristics (such as, but not limited to, resistance to herbicide, pest or disease resistance, resistance to environmental stress, modified nutrient content and improved yield) to produce progeny plants that have recombinant DNA that confers both the desired target sequence expression behavior and the trait(s)( s) additional(s). Typically, in such breeding for combining traits the transgenic plant that donates the additional trait is of male lineage and the transgenic plant carrying the base traits is of female lineage. The progeny of this cross separate such that some of the plants will carry DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with the parental recombinant DNA. Progeny plants that carry DNA for both parental traits can be backcrossed to the parental female line multiple times, for example, generally 6 to 8 generations, to produce a progeny plant with substantially the same genotype as a line. original transgenic parental line but with recombinant DNA from the other transgenic parental line. Yet another aspect of the invention is a transgenic plant cultivated from the transgenic seed of the invention. This invention contemplates transgenic plants grown directly from a transgenic seed containing recombinant DNA as well as generations of plant progeny, including natural or hybrid plant lines, made by crossing a transgenic plant grown directly from a transgenic seed. - genetic with a second plant not grown from the same transgenic Li seed. Crossbreeding may include, for example, the following steps: 15 (a) plant seeds from the first parental plant (e.g., non-transgenic or transgenic). and a second parental plant that is transgenic in accordance with the invention; (b) growing the seeds of the first and second parental plants into plants that have flowers; 20 (c) pollinate a flower from the first parent with pollen from the second parent; and (d) collect seeds produced on the parent plant that has the fertilized flower. It is often desirable to introduce recombinant DNA into elite varieties, for example, by backcrossing, to transfer a specific desirable trait from a source to a natural plant or another plant that does not have that trait. This can be achieved, for example, by first crossing a superior mating organism ("A") (recurrent parent) with a donor mating organism ("B") (non-recurrent parent), which carries the gene(s). (s) appropriate for the trait in question, for example, a construct prepared in accordance with the present invention The progeny of such a cross is first selected from the resulting progeny for the desired trait to be transferred to the non-recurrent parent. "B" and then the selected progeny is backcrossed to the superior recurrent parent "A". After five or more generations of backcrossing with selection for the desired trait, the progeny are homozygous for the loci controlling the trait being transferred. , but is similar to the superior parent in most or almost all other genes. The last backcross generation can be self-fertilized to give progeny that are pure generation for the gene(s) being transferred. , that is, one or more transformation events.

Through a series of breeding manipulations, a selected DNA construct can be passed from one strain to an entirely different strain without the need for additional recombinant manipulation. One can thus produce natural plants that are true generations for one or more DNA constructs. By crossing different natural plants, a large number of different hybrids with different combinations of DNA constructs can be produced. In this way, plants can be produced that have the desirable agronomic properties often associated with hybrids ("hybrid vigor"), as well as the desirable characteristics transmitted by one or more DNA constructs.

Genetic markers can be used to assist in introducing one or more DNA constructs of the invention from one genetic background to another. Marker-aided selection offers advantages over conventional breeding because it can be used to avoid errors caused by phenotypic variations. Furthermore, genetic markers can provide data regarding the relative degree of elite germplasm in the individual progeny of a particular cross. For example, when a plant with a desired trait that otherwise has a non-agronomically desirable genetic background is crossed with an elite parent, genetic markers can be used to select progeny that not only possess the trait of interest, but also have a relatively large proportion of the desired germplasm. In this way, the number of generations required to introduce one or more characteristics into a particular genetic background is minimized. The utility of marker-aided selection in crossing transgenic plants of the present invention, as well as types of useful molecular markers, such as, but not limited to, SSRs and SNPs, is discussed in PCT Application Publication WO 02/062129 and in Publication Applications of 5 U.S Patent Numbers 2002/0133852, 2003/0049612 and 2003/0005491.

In certain transgenic plant cells and transgenic plants of the invention, it may be desirable to concomitantly express (or suppress) a gene of interest while also regulating the expression of a target gene. Thus, in some embodiments, the transgenic plant contains the recombinant DNA further including a gene expression (or suppression) element to express at least one gene of interest, and regulation of target gene expression is preferably effected with concomitant expression (or suppression) of at least one gene of interest in the transgenic plant.

Thus, as described herein, the transgenic plant cells or transgenic plants of the invention can be obtained by use of any appropriate transient or stable integral or non-integral transformation method known in the art or described at the time. The recombinant DNA constructs can be transcribed in any cell or plant tissue or in a complete plant of any stage of development. Transgenic plants may be derived from any monocotyledonous or dicotyledonous plant, such as, but not limited to, plants of commercial or agricultural interest, such as crop plants (especially crop plants used for human food or animal feed), trees that produce wood or pulp, vegetables, fruit plants and ornamental plants. Non-limiting examples of plants of interest include grain crop plants (such as wheat, oats, barley, corn, rye, triticale, rice, millet, sorghum, quinoa, amaranth, buckwheat); forage crop plants (such as forage grass and forage dicotyledons including alfalfa, vetch, clover and the like); cultivation of oilseed plants (such as cotton, saffron, sunflower, soybeans, canola, rapeseed, linseed, peanuts and palm oil); nut trees (such as walnut, cashew, hazelnut, pecan, almond and the like); sugar cane, coconut, date palm, olives, tea and coffee; trees that produce wood and pulp; vegetables such as legumes (e.g., beans, peas, lentils, alfalfa, peanuts), lettuce, asparagus, artichokes, celery, carrots, horseradish, brassicas (e.g., cabbage, kale, mustard greens and other leafy brassicas, broccoli, cauliflower, Brussels sprouts, turnips, kohlrabi), edible cucurbits (cucumbers, melons, zucchini, pumpkin), edible garlics (e.g. onions, garlic, leeks, shallots, chives), edible members of the Solanaceae (e.g., tomatoes, eggplants, potatoes, peppers, blackberries) and edible members of the Chenopodiaceae (e.g., beets, chard, spinach, quinoa, amaranth); fruit crop plants such as apples, pears, citrus fruits (e.g. orange, lime, lemon, grapefruit and others), stone fruits (e.g. apricot, peach, plum, nectarine), banana, pineapple, grapes, kiwi, papaya, avocado and cherries; and ornamental plants including ornamental flowering plants, ornamental trees and shrubs, ornamental ground covers and ornamental grasses. Preferred dicotyledonous plants include, but are not limited to, canola, cotton, potatoes, quinoa, amaranth, buckwheat, safflower, soybeans, beets and sunflowers, more preferably soybeans, canola and cotton. Preferred monocots include, but are not limited to, wheat, oats, barley, corn, rye, triticale, rice, ornamental and forage grasses, sorghum, millet and sugar cane, more preferably corn, wheat and rice. The main objective in plant transformation is to produce plants that are useful to humans. In this regard, the transgenic plants of the invention can be used for virtually any purpose considered to be of value to the farmer or consumer. For example, one may wish to harvest the transgenic plant itself or harvest the transgenic seed from the transgenic plant for planting purposes or products may be made from transgenic plants or their seeds, such as oil, starch, ethanol or other fermentation products. , animal feed or human food, pharmaceuticals and various industrial products. For example, corn is used extensively in the food and feed industry, as well as in industrial applications. Additional discussion of the uses of corn can be found, for example, in U.S. Patent Numbers 6,194,636, 6,207,879, 6,232,526, 6,426,446, 6,429,357, 6,433,252, 6,437,217, and 6,583,338 and in PCT Publications WO 95/06128 and WO 02/057471. Thus, this invention also provides consumer products produced from a transgenic plant cell, transgenic plant or seed of this invention, including, but not limited to leaves, roots, shoots, tubers, stems, fruits, seeds or other parts of a plant, cereals, oils, extracts, products of fermentation or digestion, crushed or whole grains or seeds of a plant or any food or non-food product including such consumer products produced from a plant cell, plant or seed transgenic version of this invention. The detection of one or more nucleic acid sequences of the recombinant DNA constructs of this invention in one or more consumer products contemplated herein is in fact evidence that the consumer product contains or is derived from a plant cell, plant or transgenic seed of this invention.

In preferred embodiments, the transgenic plant that has in its genome a recombinant DNA construct of this invention has at least one additional altered characteristic, relative to a plant that does not have the recombinant DNA construct, selected from the group of characteristics consisting of in: (a) improved tolerance to abiotic stress; (b) improved tolerance to biotic stress; (c) modified primary metabolite composition; (d) modified secondary metabolite composition; (e) trace element, carotenoid or modified vitamin composition; (f) improved yield; (g) improved ability to use nitrogen or other nutrients; (h) modified agronomic characteristics; (i) modified growth or reproductive characteristics; and Q) improved harvesting, storage and processing quality.

In particularly preferred embodiments, the transgenic plant is characterized by: improved tolerance to abiotic stress (e.g., tolerance to lack of water or drought, heat, cold, non-optimal nutrient or salt levels, non-optimal light levels) or from biotic stress (e.g. crowding, allelopathy or injury); by a modified primary metabolite composition (e.g., fatty acid, oil, amino acid, protein, sugar or carbohydrate); modified secondary metabolite composition (e.g. alkaloids, terpenoids, polyketides, non-ribosomal peptides and secondary metabolites of mixed biosynthetic origin); modified trace element (e.g., iron, zinc), carotenoid (e.g., beta-carotene, lycopene, lutein, zeaxanthin or other carotenoids and xanthophylls) or vitamin (e.g., tocopherols) composition; improved yield (e.g., improved yield under non-stress conditions or improved yield under biotic or abiotic stress); improved ability to use nitrogen or other nutrients; modified agronomic traits (e.g., delayed maturation, delayed senescence; early or delayed maturity; improved shade tolerance; improved resistance to root or stem conditioning; improved resistance to stem green snap; modified light photoperiod response); modified growth or reproductive characteristics (e.g., intentional dwarfism; intentional male sterility, useful in, for example, improved hybridization procedures; improved vegetative growth rate; improved germination; improved male or female fertility); improved harvesting, storage and processing quality (e.g. improved resistance to pests during storage, improved resistance to breakage, improved attractiveness to consumers); or any combination of these characteristics.

In a preferred embodiment, transgenic seed or seed produced by the transgenic plant, has modified primary metabolite composition (e.g., fatty acid, oil, amino acid, protein, sugar or carbohydrate), modified secondary metabolite composition (e.g., alkaloids , terpenoids, polyketides, non-ribosomal peptides and secondary metabolites of mixed biosynthetic origin), trace element composition (e.g. iron, zinc), carotenoid (e.g. beta-carotene, lycopene, lutein, zeaxanthin or other carotenoids and xanthophylls) or modified vitamin (e.g. tocopherols), improved harvesting, storage or processing quality or a combination thereof. For example, it may be desirable to modify the amino acid (e.g., lysine, methionine, tryptophan, or total protein), oil (e.g., fatty acid composition or total oil), carbohydrate (e.g., simple sugars or starches), trace element, carotenoid or vitamin from seeds of cultivable plants (e.g. canola, cotton, safflower, beetroot, sunflower, wheat, maize or rice), preferably in combination with improved seed harvest, storage and processing quality and thus provide improved seeds for use in animal feed and human food. In another example, it may be desirable to alter the levels of native components of the transgenic plant or transgenic plant seed, for example, to decrease the levels of proteins with low levels of lysine, methionine or tryptophan or to increase the levels of an amino acid or acid. desired fatty acids or to decrease levels of an allergenic protein or glycoprotein (e.g., peanut allergens including ara h 1, wheat allergens including gliadins and glutenins, soy allergens including P34 allergen, globulins, glycinins and conglycinins) or of a toxic metabolite (e.g., cyanogenic glycosides in cassava, solanine alkaloids in members of the Solanaceae).

EXAMPLES Example 1

This illustrates non-limiting examples of recombinant DNA constructs for suppressing at least one target gene; These constructs, when designed to include a miRNA recognition site, allow expression of the gene suppression element in specific tissues. Figure 1A schematically depicts non-limiting examples of recombinant DNA constructs of the invention for suppressing at least one target gene. These constructs include at least one first gene suppression element ("GSE" or "GSE1") for suppressing at least one first target gene, wherein the first gene suppression element is inserted into an intron flanked on one or both sides by a DNA that does not code for protein. These constructs utilize an intron (in many embodiments an intron derived from a 5' untranslated region or an intron that enhances expression is preferred) to release a gene suppression element without requiring the presence of any protein-coding exons (coding sequence). ). The constructs may optionally include at least one second gene suppression element ("GSE21') to suppress at least one second target gene, at least one gene expression element ("GEE") to express at least one gene of interest (which may be a coding or non-coding sequence or both), or both. In embodiments that contain an optional gene expression element, the gene expression element may be located outside (e.g., adjacent to) the intron. intron containing the first gene suppression element is 3' to a terminal dor. To more clearly differentiate recombinant DNA constructs of the invention (which contain at least one gene suppression element inserted within a single intron flanked in one or both of the sides by a non-protein-coding DNA) of the prior art, Figure 20 1B schematically describes examples of prior art DNA constructs. These constructs may contain a gene suppression element that is located adjacent to an intron flanked by a coding sequence. of protein or between two distinct introns (in which the gene suppression element is not inserted into either of the two distinct introns) or may include a gene expression element that includes a gene suppression element inserted within an intron that is flanked by multiple exons (e.g., exons that include the coding sequence of a protein).

Figure 2 describes several non-limiting examples of gene suppression elements and exogenous DNAs capable of being usefully transcribed into the recombinant DNA constructs of the invention. Where drawn as a single strand (Figures 2A to 2E), these are conventionally depicted in the 5' to 3' (left to right) transcriptional direction; the arrows indicate the antisense sequence (arrowhead pointing to the left), or the sense sequence (arrowhead pointing to the right). Such gene suppression elements and exogenous DNAs capable of being transcribed may include: DNA that includes at least one antisense DNA segment that is antisense to at least one segment of the at least one first target gene, or DNA that includes multiple copies of at least one antisense DNA segment that is antisense to at least one segment of the at least one first target gene (Figure 2A); DNA that includes at least one sense DNA segment that is at least one segment of the at least one first target gene, or DNA that includes multiple copies of at least one sense DNA segment that is at least one segment of the at least one first target gene (Figure 2B); DNA that transcribes RNA will suppress the at least one first target gene by forming double-stranded RNA and includes at least one antisense DNA segment that is antisense to at least one segment of the at least one target gene and at least at least one segment of sense DNA that is at least one segment of the at least one first target gene (Figure 2C); DNA that transcribes RNA to suppress the at least one first target gene by forming a single double-stranded RNA and includes multiple serial segments of antisense DNA that are antisense to at least one segment of the at least one first target gene and multiple sense serial DNA segments that are at least one segment of at least one first target gene (Figure 2D); DNA that transcribes RNA to suppress at least one first target gene by forming multiple double-stranded RNA and includes multiple antisense DNA segments that are antisense to at least one segment of the at least one first target gene and multiple segments of sense DNA having at least one segment of the at least one first target gene and wherein said multiple segments of antisense DNA and said multiple segments of sense DNA are arranged in a series of inverted repeats (Figure 2E ); and DNA that includes nucleotides derived from a miR NA or DNA that includes nucleotides from an siRNA (Figure 2F). Figure 2F depicts various non-limiting arrangements of double-stranded RNA that can be transcribed from embodiments of the gene suppression elements and exogenous DNAs capable of being usefully transcribed into the recombinant DNA constructs of the invention. When such double-stranded RNA is formed, it can suppress one or more target genes, and can form a single double-stranded RNA or multiple double-stranded RNA, or a single stem or multiple stems of double-stranded RNA. Where multiple double-stranded RNA "stems" are formed, they may be arranged in "hammerhead" or "cloverleaf" arrangements. In some embodiments, the double-stranded stems may form a "pseudo-knot" arrangement (e.g., when the spacer or loop RNA from one double-stranded stem forms part of a second double-stranded stem). pair); see, for example, descriptions of pseudo-node architectures in Staple & Butcher (2005) PLoS Biol., 3(6):e213. Spacer DNA (located between or adjacent to dsRNA regions) is optional but commonly included and generally includes DNA that does not correspond to the target gene (although in some embodiments it may include sense or antisense DNA of the target gene). Spacer DNA may include sequence that transcribes single-stranded RNA or at least partially double-stranded RNA (such as in a "kissing stem-loop" arrangement) or an RNA that assumes a secondary structure or three-dimensional configuration (e.g., a large loop antisense sequence of the target gene or an aptamer) that confers on the transcript an additional desired characteristic, such as increased stability, increased in vivo half-life, or cell or tissue specificity.

Example 2

This example describes a non-limiting method of determining the potential utility of a miRNA recognition site by determining the pattern of miRNA expression. Knowledge of the spatial or temporal distribution of a given miRNA expression is useful, for example, when designing recombinant constructs to be expressed in a spatially or temporally specific manner. This example describes mature miRNA expression patterns in maize and provides recognition site sequences for these miRNAs that are suitable for inclusion in recombinant DNA constructs useful in maize and other plants. Total RNA was isolated from LH244 corn plants using Trizol (Invitrogen, Carlsbad, CA). Seven developmental stages were used, including root and branch meristems of germinating seedlings, young (V1 to V2) and adult (V7 to V8) leaves, internodal stem, tassel before fall, and immature ears (approximately 1") (2.54 cm). Five micrograms of total RNA were separated on PAGE-17% Urea as described by Allen et al. (2004) Nat. Genet., 36:1282-1290. Blots were labeled with DNA oligonucleotides that were antisense to the small RNA sequence and end labeled with gamma 32P-ATP using Optikinase (USB). The probes used and their respective sequences are given in Table 1. Table 1. The results are shown in Figure 3. A probe (SEQ ID NO: 1) hybridized with mature miRNAs from two families (miR156 and 15 mÍR157). Individual mature miRNAs were expressed at different levels in specific cells or tissues. For example, Zm-miR390 was not expressed or expressed only at low levels in adult root and leaf.

Example 3

This non-limiting example describes recombinant DNA constructs of the invention useful for suppressing the expression of a target RNA in a specific cell of or derived from a multicellular eukaryote such as a plant cell or an animal cell and methods for their use. - 5 so. The constructs include a promoter operably linked to DNA that transcribes RNA including at least one exogenous miRNA recognition site recognizable by a mature miRNA expressed in a specific cell of a multicellular eukaryote, and a target RNA to be suppressed in the specific cell, wherein said target RNA is to be expressed in cells of the multicellular eukaryote other than the specific cell. Strong constitutive promoters that are expressed in approximately all plant cells have been identified (e.g., CaMC 35S, OsAct), but spatially specific (cell or tissue specific) and temporally specific strong promoters have been less well characterized. To limit the expression of target RNA or transgene to a specific cell or tissue type in the absence of a strong cell- or tissue-specific promoter, it may be desirable to suppress in selected cells or tissues the expression of a transcript under the control of a promoter. constitutive. The invention provides methods that use 20 endogenous miRNA recognition sequences to suppress the expression of a target RNA constitutively expressed in specific cells.

Methods of the invention allow spatially or temporally specific post-transcriptional control of expression of a target RNA in which transcription is directed by a non-specific promoter (e.g., constitutive). The methods of the invention allow, for example, restricted expression of a gene transcribed by a constitutive promoter or a promoter with expression beyond the desired cell or tissue type(s). Restricted expression may be restricted spatially or temporally, for example, restricted to specific types or classes of tissues or cells or to specific developmental, reproductive, growth, and seasonal stages. Where a miRNA is expressed under particular conditions (e.g. under biotic stress such as crowding, allelopathic interactions or pest or pathogen infestation, or abiotic stress such as heat or cold stress, drought stress, nutritional stress, heavy metal or salt), the corresponding miRNA recognition site can be used for conditionally specific suppression, that is, to suppress the target RNA under the particular condition. For example, Zm-miR162 is poorly expressed in maize roots (see Example 2 and Figure 3), therefore, designing an expression construct to include an exogenous miRNA162 recognition site adjacent to, or within, a constitutively expressed target RNA , can limit the accumulation of target RNA transcripts in all cells of a corn plant except the roots. This method has utility for all 1.1 gene expression applications in which the given mature miRNA is expressed.

In multicellular eukaryotes, including plants, microRNAs (miRNAs) regulate endogenous genes by a post-transcriptional cleavage mechanism, which can be spatially or temporally specific. The present invention provides methods by which the addition of a miRNA recognition site to a constitutively expressed transgene could be used to limit expression of the transgene to cells that do not have, or are distant from those that express, the complementary mature miRNA both spatially and spatially. temporally (including conditionally). Manipulation of these miRNA recognition sites on new transcripts introduced into transgenic plant cells, and transgenic plants derived from these cells, is useful for altering expression patterns for the new transgene.

In an alternative approach, an existing miRNA recognition site (native or endogenous) is mutated (e.g., by chemical mutagenesis) sufficiently to reduce or prevent cleavage (see Mallory et al. (2004) Curr. Biol. , 14:1035-1046). In this way, a target RNA sequence with desirable effects, for example, increased leaf or seed size, can be expressed at higher levels than when the native or endogenous miRNA recognition site was present. One modality is to replace a native gene with an engineered homologue, in which the native miRNA has been mutated or even deleted, that is, it is less susceptible to cleavage by a given miRNA.

One embodiment of the method is the introduction of at least one exogenous miRNA recognition site (typically a 21-nucleotide sequence) into the 5' or 3' untranslated regions of a target RNA, or within the target RNA. Where the target RNA includes the coding sequence, the at least one exogenous miRNA recognition site can be introduced into the coding region of the target RNA. This results in reduced expression of the target RNA in tissue or cell types that express the corresponding mature miRNA. By including a recognition site that corresponds to a mature miRNA in a target RNA transcript, it is possible to modulate the expression of the target RNA in such a way that even under the control of a constitutive promoter, the target RNA is expressed only in cells or selected fabrics or during selected time periods. This allows for both the high levels of expression obtainable with strong constitutive promoters and the spatial or temporal limitation of such expression. Any miRNA recognition site can be used, preferably where expression of the corresponding mature miRNA has been determined to satisfy the desired expression or suppression of the target RNA. Several miRNA recognition sequences are known. See, for example, Jones-Rhoades & Bartel (2004). Mol. Cell, 14:787-799, Rhoades et al. (2002) Cell, 110:513-520, Allen et al. (2004) Nat. Genet., 36:1282-1290). See also the database on the ASRP network (Gustafson et al. (2005) Nucleic Acids Res., 33:D6379-D640). Non-limiting examples of miRNA recognition sites useful in constructs and methods of the invention include those provided in Table 2, which gives the recognition site sequences for the indicated miRNA family and indicates the distribution among "all plants" (i.e., lower plants, monocotyledons and dicotyledons), monocotyledons and/or dicotyledons The plant species from which the miRNA was identified and the abbreviations used were: A- rabidopsis thaliana (At), Glycine max (Gm), Gossypium hirsutum (Gh). ), Hordeum vulgare (Hv), Lycopersicum esculentum (Le), Lotus comiculatus var. , Pennisetum glaucum (Pg), Phaseolus vulgaris (Pv), Populus tremula (Pt), Saccharum officina- rum (So), Sorghum bicolor (Sb), Theobroma cacao (To), Triticum aestivum (Ta), Vitis vinifera ( Vv), and Zea mays (Zm). Table 2 Table 2 continued Table 2 (continued) Table 2 continued Table 2 continued Table 2 continued Table 2 continued

Thus, a transgenic plant that expresses a recombinant DNA construct that, under the control of a constitutive promoter (e.g., a 35S promoter) transcribes an RNA that contains a Zm-miR390 recognition site and a target RNA could it would be expected to show suppression of target RNA expression in the root and adult leaf in relation to expression in other tissues.

In another example, Zm-miR172 was expressed at high levels in the stem and not expressed, or expressed only at low levels, in other tissues. A transgenic plant that expresses a construct that, under the control of a strong constitutive promoter (e.g., a CaMV 35S promoter) transcribes an RNA that contains a Zm-miR172 recognition site and a target RNA would be expected to express that target RNA. at higher levels in tissues other than the stem (where target RNA expression would be suppressed). To illustrate the use of the constructs and methods of the invention to control the expression of a gene of interest, a reporter gene is used as the gene of interest itself or as a surrogate for the gene of interest. For example, where expression of a reporter gene (e.g., green fluorescent protein, GFP) is desired in corn stalk and immature ear tissue, a miR156 target site is included in a GFP expression cassette and expressed. in a stably transgenic maize plant under the control of the CaMV35S promoter. In other tissues (e.g., roots, leaves, and tassels), GFP expression is suppressed. The suppression phenotype may be limited to very specific cell types within the 15 suppressed tissues, with neighboring cells showing expression or a gradient of GFP expression adjacent to those cells expressing mature miR156.

In another example, a strong constitutive promoter is used to direct the expression of a Bacillus thuringiensis ("Bt") insecticidal protein or protein fragment, where a pollen-specifically expressed miRNA recognition site is included in the construct, resulting in strong expression of Bt in plant tissues except for pollen.

A specific, non-limiting example of the method is the inclusion of the recognition site for at least one miRNA, which is not expressed (or expressed only at low levels) in roots, in a recombinant DNA construct that includes a target RNA of which expression is desired only in the roots. A strong constitutive promoter (e.g., enhanced 35S) can still be used, but expression of the target RNA is now restricted to cells that do not express the corresponding mature miRNA. A specific example of this approach is the inclusion of a maize miRNA162, maize miRNA164, or maize miRNA390 recognition site (or any combination thereof) in a recombinant DNA construct for expression of a Ba insecticidal protein or protein fragment. -cillus thuringiensis ("Bt", see, for example, the insecticidal sequences of Bacillus thuringiensis and methods of use thereof described in U.S. Patent Number 6,953,835 and U.S. Provisional Patent Application Number 60/713,111, 5 filed on August 31 2005) as the target RNA, for example, in a construct that includes the e35S/Bt/hsp17 expression cassette. These miRNAs (e.g., miRNA162, miRNA164, or mRNA390) are not substantially expressed in maize roots but are expressed in most other tissues. The inclusion of one or more of these recognition sites within an expression cassette reduces transcript expression in most tissues other than the root, but maintains high levels of Bt target RNA expression. on the roots, as is desirable for controlling pests such as corn rootworm. In similar embodiments, combinations of different miRNA recognition sites are included in the construct in order to obtain the desired expression pattern in one or more specific tissues.

Specific non-limiting examples of a DNA sequence capable of being transcribed that includes an exogenous miRNA recognition site are depicted in Figure 4 and Figure 5. Figure 4 depicts chloroplast-targeted TIC809 20 with a miRNA162 recognition site (bold text ) located in the 3' untranslated region (SEQ ID NO. 176). Figure 5 depicts non-targeted TIC809 with a miRNA164 recognition site (bold text) located in the 3' untranslated region (SEQ ID NO. 177).

Example 4

This example describes the identification of a MIR gene from a crop plant with tissue-specific expression and identification of the miR gene promoter. More particularly, this example describes transcription profiling as a non-limiting method of identifying a miRNA specifically expressed in female reproductive tissue (endosperm), as well as identifying a maize miR167 promoter sequence with endosperm-specific expression. The recognition site corresponding to this miRNA is useful, for example, in the production of transgenic plants with inducible female sterility according to this invention.

A member of the MIR167 gene family (SEQ ID NO. 178) was found to represent about a quarter of the small RNA population cloned from developing maize endosperm. To determine whether a single member of the MIR167 gene family is responsible for the strong expression observed in the endosperm, several MIR167 genes were analyzed by RT-PCR. Nine stem-loop sequences of MIR167 from Zea mays were found in the public miRNA registry ("miRBase", available online at microrna.sanger.ac.uk/sequences) listed as miR167a through miR167í. Tissue-specific RT-PCR was performed for several of the Z. mays miR167 sequences using gene-specific primers for first-strand cDNA synthesis followed by PCR with gene-specific primer pairs. Expression of miR167 was strong and specific to endosperm tissue (15, 20 days after pollination). To determine whether miR167g is abundantly expressed in the endosperm, Northern blots of maize (LH59) were prepared. The blot was labeled with a mature end-labeled miR167 22-mer LNA probe (Figure 6A), stripped, and relabeled with a ~400bp gene-specific miR167g probe (Figure 6B). The strong endosperm signal observed indicated that miR167g is largely responsible for the increased expression in the endosperm. Transcriptional profiling of maize tissues confirmed the Northern blot results (Figure 6C); the transcript corresponding to miR167g was abundantly and specifically expressed in endosperm tissue.

An 804 base pair cDNA sequence publicly available in GenBank (marked as "ZM_BFb0071l20.r ZM_BFb Zea mays cDNA 5', mRNA sequence") and having the accession number DR827873.1 (Gl:71446823) is incorporated here by reference. This sequence includes a segment (SEQ ID NO. 178) that corresponds to mature miR167g. Using the public sequence, bioinformatics analysis was performed on the patented maize genomic sequence. A 4.75-kilobase genomic cluster that includes the sequence from the natural maize line B73 was identified as containing predicted gene sequences for miR167a and miR167g. A 486 base pair region between the two miR167 genes was identified as having homology to an expressed sequence flag sequence (EST). Promoter motifs were identified in the upstream sequences of both predicted transcripts (miR167a and miR167g). A region of 1682 base pairs (SEQ ID NO. 179) between the predicted miR167a and miR167g transcripts, and a smaller region of 674 base pairs (SEQ ID NO. 180) between the EST and the predicted miR167g transcript were identified. - each as miR167g promoter sequences. Subgroups of these sequences (e.g., at least about 50, about 100, about 150, about 1.1 of 200, about 250, or about 300 nucleotides of SEQ ID NO. 179 or SEQ ID NO. 180 or fragments of at least about 50, about 100, about 150 and about 200 contiguous nucleotides that have at least 15 85%, at least 90% or at least 95% identity to a segment of SEQ ID NO 179. or SEQ ID NO. 180) are also useful as promoters; its promoting effects are demonstrable by procedures well known in the art (for example, to direct the expression of a reporter gene such as luciferase or green fluorescent protein). The annotation map, including gene locations of miR167a and miR167g and mature miRNAs and promoter elements (e.g., TATA boxes), of this genomic cluster is shown in Figure 7. The annotation map also shows the location of motifs of auxin responsive factor (ARF) or auxin response elements with the sequence TGTCTC (SEQ ID NO. 181), which indicates that auxin can regulate the expression of miR167g. Mature miR167 miRNAs are complementary to ARF6 and ARF8 (which encode activating ARFs) and have been proposed to regulate auxin homeostasis; see, for example, Rhoades etal. (2002) Cell, 110:513-520, Bartel & Bartel (2003) Plant Physiol., 132:709-717, Ulmasov et al. (1999) Proc. Natl. Academic. Sci. USA, 96:5844-5849, and Mallory et al. (2005) Plant Cell, 17:1360-1375.

In addition to the miR167g promoter sequences (SEQ ID NO. 179 and SEQ ID NO. 180) identified from the natural maize line B73, two additional miR167g promoter sequences (SEQ ID NO. 182 and SEQ ID NO. 183) were amplified from from the natural corn line LH244. The 3' ends of SEQ ID NO. 182 and SEQ ID NO. 183 were determined experimentally by 5'RACE (rapid amplification of cDNA ends, Invitrogen Corporation, Carlsbad, CA) of miR167g. The 5' end of the 768 base pair sequence (SEQ ID NO. 182) corresponds to the end of a 481 base pair DNA sequence publicly available in GenBank (annotated as "QCG17cO3.yg QCG Zea mays cD - NA clone QCG17cO3, mRNA sequence") and which has the accession number CF035345.1 (GI:32930533). The 5' end of the 407 base pair sequence (SEQ ID NO. 183) corresponds to the end of a 746 base pair DNA sequence publicly available in GenBank (annotated as "MEST991„A06.T7-1 UGA-ZmSAM- XZ2 Zea mays cDNA, mRNA sequence") and which has the accession number DN214085.1 (GI:60347112).

The miR167g promoter sequences, míR167g gene, mature miR167g microRNA and miR167g recognition site (examples of which are included below in Table 6) described here have several uses as described elsewhere in this description. In particular, a miR167g promoter is useful as an endosperm-specific promoter and can be used, for example, to replace a maize B32 promoter. In another use, the sequence of miR167g or mature miR167g (or a precursor thereof) is manipulated to suppress a target gene, especially when the suppression must be endosperm specific. The miR167g recognition site is useful, for example, in constructs for gene expression where the gene must be expressed in tissues other than the endosperm and especially useful in the production of transgenic plants with inducible female sterility according to this invention.

Example 5

Current criteria for miRNA identification have emphasized the phylogenetic conservation of miRNAs across species, and thus some non-conserved or species-specific miRNAs in plants have been characterized in plants. This example describes non-limiting methods for identifying miRNAs, their tissue expression patterns, their corresponding recognition sites, and example targets. Five novel non-conserved miRNAs and corresponding MIR sequences were identified from a size-fractionated c-DNA library constructed from soybean leaves. Criteria for miRNA identification included: (1) a cloned 21-nt small RNA clone, and a putative miRNA* (strand corresponding to the miRNA) at a lower abundance, (2) containment of the miRNA/miRNA* duplex entirely within a small, imperfect foldback structure, (3) derivation of the 10 miRNA from a non-protein-coding RNA Pol II transcript and (4) presence of a complementary target site in a coding gene; see 111 Ambros et al. (2003) RNA, 9:277-279. Small RNAs were extracted from raw sequences containing an adapter and their strands were determined. This group of 15 sequences was filtered to remove small RNA sequences that were virus RNA, tRNA, rRNA, chloroplast and mitochondrial RNA, and transgene, resulting in a filtered group of 381,633 putative miRNA sequences. Small RNAs that do not originate from the above sources and are not homologous to known miRNAs were mapped against soybean cDNA sequences. For mapped cDNA sequences with low protein-coding content, a cDNA sequence fragment of about 250 nucleotides, containing the putative miRNA, was folded using RNA Folder. The foldback structure was examined to see if the small RNA was located in 'stem11, and if an extensively (but not perfectly) complementary small RNA with lower abundance was located on the opposite side of 'stem'1. Potential small RNA targets are predicted based on rules modified from Jones-Rhoades & Bartel (2004) Mol. Cell, 14:787-799, and Zhang (2005) Nucleic Acids Res., 33:W701-704. Table 3 lists the five novel non-conserved miRNAs cloned from soybean leaf tissue and for each, corresponding miRNA* and primRNA(s); Abundance ("abundance") is given as the number of times the sequence occurred out of a total of 381,633 sequences. Table 3 For each new soybean miRNA, the foldback structure of the miRNA precursor sequences was predicted by an RNAfold-based algorithm (RNAFolder), publicly available at www.tbi.univie.ac. at/~ivo /RNA/RNAfold.html), and the miRNA precursor transcription profile obtained when available, as listed in Table 4. Examples of predicted targets (recognition sites) in soybean and their i-identified expression patterns were identified for two of the miRNAs (SEQ ID NO. 184 and SEQ ID NO. 187). Table 4 Additionally, target sequences (recognition site) for each new soybean miRNA were identified from internal soybean databases ("MRTC"), as listed in Table 5. The number of unpaired nucleotides between each recognition site and the hard miRNA sequence ranges from 1 to 6 mismatches, which may include gaps or "missing" nucleotides. For example, a gap is found between nucleotides 7 and 8 in each of SEQ ID NO. 220 , SEQ ID NO. 221, SEQ ID NO. Table 5 continued Table 5 continued Table 5 continued Table 5 continued

Example 6

This example describes non-limiting methods for identifying miRNAs for use in the production of transgenic plants with inducible sterility usable in methods of this invention. But specifically, 5 miRNAs that have an expression pattern in the desired tissues (i.e., male or female reproductive tissues) were identified by evaluating transcriptional profiling (TxP) data.

A common procedure for identifying miRNAs that have a pattern of expression in desired tissues (i.e., male or female reproductive tissues) includes the steps of: (a) providing a starting miR sequence that includes the region *■' "stem- loop", for example, from publicly available miR sequences in the "miRBase" database (available online at microrna.sanger.ac.uk/sequences); (b) apply sequence analysis algorithms, such as

BLAST as is well known in the art (see Altschul et al. (1990) J. Mol. Biol., 215:403-410) to identify homologous or identical sequences (e.g., patented sequences in probesets). ) microarrays made with DNA from the complete corn genome); and (c) analyze the transcription profiles of the homologous sequences from the probe set identified in step (b) and identify miRNAs that have an expression pattern in the desired 25 tissues (i.e., male or female reproductive tissues). Preferably, a fourth step is added: (d) for discovered homologous probe set sequences to have the desired transcriptional profiles, confirming identification of the miRNA gene by both stem-loop sequence alignment of the initial miR with the sequence of the probe set, as well as by miR-

Potentially new NAs, determine the sequence that is predicted to fold into a stem-loop structure characteristic of a miRNA. Also preferably, an optional step is used, in which one or more BLAST comparisons against data sets of additional sequences different from the probe set sequence data groups are included (prior to step (b) above), allowing additional identification of probes that fall outside the predicted foldback region of the miR gene; for example, due to mismatches in additional sequence data groups that include contigs that have undergone incorrect splicing, they are identified by their lack of miRNA characteristics such as appropriate foldback structure, and removed. patented maize transcription profile. Figures 13 and 14 describe the transcription profiles of probe set sequences that have been identified as including miRNA genes that have the desired expression patterns, that is, in female reproductive tissue. (Figure 13) and in male reproductive tissue (Figure 14). Several miRNA genes and examples of their corresponding recognition sites have been identified to be useful in transgenic plant production with inducible sterility (Table 6). Two members of the miR166 family, miR166b and miR166d, shared an identical mature miRNA sequence and corresponding recognition sites, but showed different expression patterns, with miR166b being expressed only in embryo and miR166d being expressed in ovule, embryo, stigma and root; thus, to make plants inductively female sterile, miR166b would be preferred. A particularly preferred embodiment of a miRNA useful for producing inductively female-sterile plants is maize endosperm-specific miR167g (SEQ ID NO. 307), with or without additional G at the 3'end; embodiments that have additional G at the 3' end are 22-mers ending in GG and have corresponding recognition sites that are also preferably 22-mers where the nucleotide added to the 5' end is preferably unpaired (i.e., not is C). In addition to the utility of these and similar miRNAs and their corresponding recognition sites for the production and use of transgenic, inductively sterile plants, the promoters of these mature miRNAs are useful for directing the expression of a transgene for which specific expression in a reproductive tissue is desired, for example, the miR167g promoter is useful for directing specific expression in endosperm. Table 6 Table 6 continued

Example 7

This is a non-limiting example of methods for making a recombinant DNA construct useful in making an inductively sterile, transgenic plant that transcribes RNA that includes: (a) at least one exogenous miRNA recognition site recognizable by a mature miRNA that is specifically expressed in plant reproductive tissue; and (b) messenger RNA encoding a protein that confers tolerance to a herbicide; in which the mature miRNA specifically suppresses expression of the protein in reproductive tissue and in which the sterility of the transgenic plant is inducible by application of the herbicide to the plant. A non-limiting example of a method for designing a miRNA recognition site for use in such a recombinant DNA construct is illustrated here, in which the plant is corn and the protein that confers tolerance to a herbicide is 5-enolpyruvyl-shikimate-3 -phosphate synthase; Analogous methods are useful with other plants and other proteins.

This method includes the steps of: (a) Selecting a unique target sequence of at least 18 nucleotides specific for the messenger RNA encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). 20 One approach is to use a BLAST search (e.g., of both cDNA and maize genomic DNA databases) to identify EPSPS orthologs and any potential matches to unrelated genes, thereby avoiding unintentional silencing of non-target sequences. (b) Analyze the messenger RNA for undesirable sequences (e.g., pairings with sequences from non-target species, especially animals) and score each potential 19-mer segment for GC content, Reynolds score 30 (see Reynolds et al (2004) Nature Biotechnoi., 22:326-330), and functional asymmetry characterized by a negative difference in free energy ("ΔΔG") (see Khvorova etal. (2003) Cell, 115:209-216) . Preferably 19-mers are selected for having all or most of the following characteristics: (1) a Reynolds score >4, (2) a GC content between about 40% to about 60%, (3) a negative MG , (4) a terminal adenosine, (5) lack of a consecutive series of 4 or more of the same nucleotides; (6) a location close to the 3' end of the target gene. Preferably, multiple 19-mers (3 or more) are selected for testing. (c) Determine the reverse complement of the 19-mers selected for use in the production of a synthetic 21-mer miRNA; the additional nucleotide at position 20 is preferably paired with the selected target sequence and the nucleotide at position 21 is preferably chosen to be unpaired to prevent transience. (d) Test synthetic miRNAs, for example, in a Nicotiana benthamiana transient assay for miRNA expression and target repression. (e) Cloning the most effective miRNAs into a construct for stable maize transformation (see the sections under the headings "Making and Using Recombinant DNA Constructs" and "Making and Using Transgenic Plant Cells and Transgenic Plants" ).

Example 8

This example describes the production of transgenic, inductively sterile plants and their uses in hybrid seed production. More specifically, this example describes the production of hybrid corn seed from an inductively male sterile transgenic corn parent plant and an inductively female sterile transgenic corn parent plant, wherein sterility is induced by the application of glyphosate herbicide. to the parental plants.

Recombinant DNA constructs are designed to transcribe (a) an exogenous miRNA recognition site recognizable by a mature miRNA that is specifically expressed in plant reproductive tissue; and (b) messenger RNA that encodes a protein (5-enolpyruvylshikimate-3-phosphate synthase) that confers tolerance to a herbicide (glyphosate); in which the mature miRNA specifically suppresses expression of the protein in reproductive tissue and in which the sterility of the transgenic plant is inducible by application of the herbicide to the plant. Each recombinant DNA construct includes a sequence that transcribes the messenger RNA encoding 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium tu-mefaciens strain CP4 ("EPSPS-CP411), ATGCTTCACGGTGCAAGCAGCCGTCCAGCAACTGCTCGTAAGTCCTCTGGTCTTTCT GGAACCGTCCGTATTCCAGGTGACAAGTCTATCTCCCACAGGTCCTTCATGTTTGGA GGTCTCGCTAGC GGTGAAACTCGTATCACCGGTCTTTTGGAAGGTGAAGATGTTATC AACACTGGTAAGGCTATGCAAGCTATGGGTGCCAGAATCCGTAAGGAAGGTGATACT TGGATCATTGATGGTGTTGGTAACGGTGGACTCCTTGCTCCTGAGGCTCCTCTCGAT TTCGGTAACGCTGCAACTGGTTGCCGTTTGACTATGGGTCTTGTTGGTGTTTACGAT TTCGATAGCACTTTCATTGGTGACGCTTCTCACT AAGCGTCCAATGGGTCGTGTG TTGAACCCACTTCGCGAAATGGGTGTGCAGGTGAAGTCTGAAGACGGTGATCGTCTT CCAGTTACCTTGCGTGGACCAAAGACTCCAACGCCAATCACCTACAGGGTACCTATG GCTTCCGCTCAAGTGAAGTCCGCTGTTCTGCTTGCTGGTCCAACACCCCAGGTATC ACCACTGTTATCGAGCCAATCATGACTCGTGACCACACTGAAAAGATGCTTCAAGGT TTTGGTGCTAACCTTACCGTTGAGACTGATGCTGACGGTGTGCGTACCATCCGTCTT GAAGGTCGTGGTAAGCTCACCGGTCAAGTGATTGATGTTCCAGGTGATCCATCCTCT ACTGCTTTCCCATTGGTTGCTGCCTTGCTTGTTCCAGGTTCCGACGTCACCATCCTT AACGTTTTGATGAACCCAACCCGTACTGGTCTCATCTTGACTCTGCAGGAAATGGGT GCCGACATCGAAGTGATCAACCCACGTCTTGCTGGTGGAGAAGACGTGGCTGACTTG CGTGTTCGTTCTTCTACTTTGA AGGGTGTTACTGTTCCAGAAGACCGTGCTCCTTCT ATGATCGACGAGTATCCAATTCTCGCTGTTGCAGCTGCATTCGCTGAAGGTGCTACC GTTATGAACGGTTTGGAAGAACTCCGTGTTAAGGAAAGCGACCGTCTTTCTGCTGTC GCAAACGGTCTCAAGCTCAACGGTGTTGATTGCGATGAAGGTGAGACTTCTCTCGTC GTGCGTGGTCGTCCTGACGGTAAGGGTCTCGGTAACGCTTCTGGAGC AGCTGTCGCT ACCCACCTCGATCACCGTATCGCTATGAGCTTCCTCGTTATGGGTCTCGTTTCTGAAAACCCTGTTACTGTTGATGATGCTACTATGATCGCTACTAGCTTCCCAGAGTTCATG GATTTGATGGCTGGTCTTGGAGCTAAGATCGAACTCTCCGACACTAAGGCTGCTTGA (SEQ ID NO. 344). For cloning convenience, each recombinant DNA construct also includes a spacer sequence in the 3' untranslated region (3'UTR) located between the messenger RNA and a terminator, TgagctcAAGAATTCGAGCTCGGTACCggatccTCTAGCTAG (SEQ ID NO. 345, with the Saci restriction sites and BamHI indicated by lowercase letters), which allows the insertion of a miRNA recognition site between the Saci and BamHI restriction sites. The DNA construct for inducible male sterility includes the sequence TqaqctcAAGAATTCGAAGACAATGCGATCCCUUUGGAGCTCGGTACCqqa tccTCTAGCTAG (SEQ ID NO. 346, with the Saci and Ba-5 mHl restriction sites indicated by lowercase letters), which contains a synthetic miRNA recognition site (indicated by underlined text), made to be recognized by a miR393, which is highly expressed in pollen (see Example 6) and is a member of a relatively small miRNA family (reducing the possibility that other members of the same miRNA family 10 suppress EPSPS-CP4 in tissues other than pollen). The DNA construct for inducible female sterility includes the sequence TqaqctcAAGAATTCGATCCGCTTTCTCTCTTCTGTCAGCTCGGTACCqqatc cTCTAGCTAG (SEQ ID NO. 347, with the Saci and Ba-15 mH! restriction sites indicated by lowercase letters), which contains a synthetic miRNA recognition site ( indicated by underlined text), made to be recognized by a miR156j, which is highly expressed in the ovule and early seed (see Example 6). This miRNA recognition site includes a single base at position 14, which can be preferably paired with a target sequence to improve specificity for miR156j recognition. Pairing at the 3' end of the miRNA is reduced (relative to endogenous targets), improving specificity for miR156j recognition. This reduces the possibility that other members of the miR156 family suppress EPSPS-CP4 in tissues other than the ovule and early seed. Each of the two recombinant DNA constructs (for inducible male sterility and inducible female sterility) is individually transformed into corn as described in "Making and Using Transgenic Plant Cells and Transgenic Plants". Briefly, a vector for plant transformation is prepared for each of the recombinant DNA constructs as described in "Making and Using Recombinant DNA Constructs" and used to transform corn plant tissue using Agrobacterium-mediated transformation and glyphosate selection. Transgenic plants from each transgenic insertion event are grown and progeny seeds are produced by self-pollination or routine crossing (as described in "Making and Using Transgenic Plant Cells and Transgenic Plants"). The progeny seeds optionally include other recombinant DNA to confer additional traits (for example, in the case of transformed plants, traits including herbicide resistance, pest resistance, tolerance for cold germination, tolerance to lack of water and the like). From these progeny seeds, transgenic seeds are selected to be cultivated in a first parental plant (which contains in its genome the recombinant DNA construct for inducible male sterility) and transgenic seeds to be cultivated in a second parental plant ( which contains in its genome the recombinant DNA construct for inducible female sterility). A mixed plant is made in the field, with a ratio of transgenic corn seed that has the construct for inducible male sterility to transgenic seed that has the construct for inducible male sterility between about 1:4 to about 1:10. The transgenic corn seed is allowed to germinate, thereby providing the first parental plant with inducible male sterility (i.e., parental plants to be pollinated) and the second parental plant with inducible female sterility (i.e., pollinating parental plants). At the appropriate stage(s) of plant development, the herbicide glyphosate is applied to the first and second parental plants by spraying an amount effective to induce male sterility on the first parental plants and female sterility on the second. parental plants. The seed collected 5 from the field is a hybrid seed produced from ovules of the first parental plant that were pollinated by pollen from the second parental plant. All materials and methods described and claimed herein can be made and used without undue experimentation as instructed by the above description. Although the materials and methods of this invention have been described in terms of preferred embodiments and illustrative examples, it will be clear to those skilled in the art that variations may be applied to the materials and methods described herein without disregarding the concept, spirit and scope of the invention. All such similar substitutes and modifications clear to those skilled in the art are considered to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims (6)

1. Recombinant DNA construct characterized by the fact that it transcribes to RNA comprising: (a) at least one exogenous miRNA recognition site recognizable by a mature endogenous miRNA that is specifically expressed in the reproductive tissue of a plant; and (b) a messenger RNA encoding a protein that confers tolerance to a herbicide, wherein said mature endogenous miRNA is at least one selected from the group consisting of miR156j (SEQ ID NO: 297), miR159c (SEQ ID NO : 302), miR166b (SEQ ID NO: 314), miR166d (SEQ ID NO: 314), miR167g (SEQ ID NO: 319), miR156i (SEQ ID NO: 323), miR160b-like (SEQ ID NO: 328) , miR393a (SEQ ID NO: 333), and miR396a (SEQ ID NO: 338). 2. The recombinant DNA construct of claim 1, wherein said at least one exogenous miRNA recognition site is located within at least one of: (a) a region 5' of the coding sequence of said first RNA messenger; (b) a region 3' of the coding sequence of said first messenger RNA; and (c) said first messenger RNA. 3. Recombinant DNA construct according to claim 1, characterized by the fact that it comprises at least one element selected from: (a) a plant promoter; (b) a gene suppression element; (c) an intron; (d) a gene expression element; (e) DNA that transcribes an RNA aptamer capable of binding a ligand; (f) DNA that transcribes an RNA aptamer capable of binding a ligand and DNA that transcribes a regulatory RNA capable of regulating the expression of a target sequence, wherein said regulation is dependent on the conformation of said regulatory RNA, and the said conformation of said regulatory RNA is allosterically affected by the binding state of said RNA aptamer; and (g) at least one T-DNA border. 4. Recombinant DNA construct according to claim 1, characterized by the fact that said tolerance-conferring protein is at least one protein selected from the group consisting of a 5-enolpyruvylshikimate-3-phosphate synthase, the 5- enolpyruvylshikimate-3- phosphate synthase from Agrobacterium tumefaciens strain CP4, glyphosate oxidoreductase, glyphosate acetyltransferase, glyphosate decarboxylase, pat, bar, dicamba monooxygenase, 2,2-dichloropropionic acid dehalogenase, acetohydroxyacid synthase, acetolactate synthase, haloarylnitrilase , modified acetyl-coenzyme A carboxylase, dihydropteroate synthase, 32 kDa photosystem II polypeptide, anthranilate synthase, dihydrodipicolinic acid synthase, phytoene desaturase, hydroxyphenyl pyruvate dioxygenase, modified protoporphyrinogen oxidase I, and aryloxyalkanoate dioxygenase. 5. Recombinant DNA construct according to claim 1, characterized by the fact that said at least one exogenous miRNA recognition site has the nucleotide sequence of any one of SEQ ID NOS: 298-301, 303-313, 315-318 and 320-322, or a combination thereof. 6. Recombinant DNA construct according to claim 1, characterized by the fact that said at least one exogenous miRNA recognition site has the nucleotide sequence of any one of SEQ ID NOS: 324-327, 329-332, 334-337 and 339-343, or a combination thereof.